U.S. patent number 6,872,250 [Application Number 10/060,853] was granted by the patent office on 2005-03-29 for microvolume crystallization method employing multiple lumens.
This patent grant is currently assigned to Syrrx, Inc.. Invention is credited to Nathaniel E. David, Peter R. David.
United States Patent |
6,872,250 |
David , et al. |
March 29, 2005 |
Microvolume crystallization method employing multiple lumens
Abstract
A method for determining crystallization conditions for a
material, the method comprising: taking a microfluidic device
comprising one or more lumens having microvolume dimensions and a
plurality of different crystallization samples within the one or
more lumens, the plurality of crystallization samples comprising a
material to be crystallized and crystallization conditions that
vary among the plurality of crystallization samples; transporting
the plurality of different crystallization samples within the
lumens; and identifying a precipitate or crystal formed in the one
or more lumens.
Inventors: |
David; Peter R. (Palo Alto,
CA), David; Nathaniel E. (San Diego, CA) |
Assignee: |
Syrrx, Inc. (San Diego,
CA)
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Family
ID: |
25369899 |
Appl.
No.: |
10/060,853 |
Filed: |
January 29, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
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877405 |
Jun 8, 2001 |
6719840 |
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Current U.S.
Class: |
117/68; 117/20;
117/206; 23/295R; 117/70; 117/69; 117/202 |
Current CPC
Class: |
C30B
7/00 (20130101); G01N 25/147 (20130101); C30B
29/58 (20130101); B01J 2219/00274 (20130101); Y10S
117/90 (20130101); B33Y 80/00 (20141201); Y10T
117/1024 (20150115); Y10T 117/1008 (20150115); Y10T
117/10 (20150115) |
Current International
Class: |
C30B
7/00 (20060101); G01N 25/00 (20060101); G01N
25/14 (20060101); C30B 007/08 () |
Field of
Search: |
;117/68,69,70,201,202,206,927 ;422/248.1 ;23/29R |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
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WO 93/06479 |
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Apr 1993 |
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WO |
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WO 00/60345 |
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Oct 2002 |
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WO |
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Other References
Sanjoh et al., "Spatiotemporal protein crystal growth studies using
microfluidic silicon divices", Journal of Crystal Grwoth vol. 196
(1999) pp. 691-702.* .
Wilson, et al. "Control of Solvent Evaporation in Hen Egg White
Lysozyme Crystallization," Journal of Crystal of Growth, vol. 116,
1992, pp. 414-420. .
Van Der Woerd, Mark, Lab-on-a-Chip Based Protein Crystallization,
Presentation: NASA, Marshall Space Flight Center, Oct. 25, 2001 (27
pages)..
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Primary Examiner: Kunemund; Robert
Attorney, Agent or Firm: Weitz; David J.
Parent Case Text
RELATED APPLICATION
This application is a continuation in part of U.S. patent
application Ser. No. 09/877,405 filed Jun. 8, 2001, now U.S. Pat.
No. 6,719,840 which is incorporated herein by reference.
Claims
We claim:
1. A method for determining crystallization conditions for a
material, the method comprising: taking a microfluidic device
comprising one or more lumens having microvolume dimensions and a
plurality of different crystallization samples within at least one
of the lumens, the plurality of different crystallization samples
being separated from each other within the lumen by one or more
dividers, the plurality of crystallization samples comprising a
material to be crystallized and crystallization conditions that
vary among the plurality of crystallization samples; transporting
the plurality of different crystallization samples within the
lumens; and identifying a precipitate or crystal formed in the one
or more lumens.
2. The method according to claim 1 wherein transporting the
plurality of different crystallization samples within the one or
more lumens is performed by a method selected from the group
consisting of electrophoresis, electroosmotic flow and physical
pumping.
3. The method according to claim 1 wherein transporting the
plurality of different crystallization samples within the one or
more lumens is performed by electrokinetic material transport.
4. The method according to claim 1 wherein the material to be
crystallized is a macromolecule.
5. The method according to claim 1 wherein the material to be
crystallized is a protein.
6. The method according to claim 1 wherein the material to be
crystallized is a macromolecule with a molecular weight of at least
500 daltons.
7. The method according to claim 1 wherein the material to be
crystallized is selected from the group consisting of viruses,
proteins, peptides, nucleosides, nucleotides, ribonucleic acids,
deoxyribonucleic acids.
8. The method according to claim 1 wherein the material to be
crystallized contains at least two or more materials selected from
the group consisting of viruses, proteins, peptides, nucleosides,
nucleotides, ribonucleic acids, deoxyribonucleic acids, small
molecules, drugs, putative drugs, inorganic compounds, metal salts,
organometallic compounds and elements.
9. The method according to claim 1 wherein the one or more lumens
have a cross sectional diameter of less than 2.5 mm.
10. The method according to claim 1 wherein the one or more lumens
have a cross sectional diameter of less than 1 mm.
11. The method according to claim 1 wherein the one or more lumens
have a cross sectional diameter of less than 500 microns.
12. A method according to claim 1, wherein the one or more divider
are formed of an impermeable material.
13. A method according to claim 12, wherein the impermeable
material is an impermeable liquid.
14. A method according to claim 12, wherein the impermeable
material is an impermeable solid.
15. A method according to claim 1, wherein the one or more dividers
are formed of a permeable material.
16. A method according to claim 1, wherein the one or more dividers
are formed of a semimpermeable material.
17. A method according to claim 16, wherein the semipermeable
material is a gas.
18. A method according to claim 16, wherein the semipermeable
material is a liquid.
19. A method according to claim 16, wherein the semipermeable
material is a gel.
20. A method according to claim 16, wherein the one or more divders
form an interface selected from the group consisting of
liquid/liquid, liquid/gas interface, liquid/solid and
liquid/sol-gel interface.
21. A method according to claim 1, wherein the one or more dividers
are selected from the group consisting of a membrane, gel, frit,
and matrix.
22. A method according to claim 1, wherein the one or more dividers
function to modulate diffusion characteristics between adjacent
crystallization samples.
23. A method according to claim 1, wherein the one or more dividers
are formed of a semipermeable material that allows diffusion
between adjacent crystallization samples.
24. A method according to claim 1, further including forming the
plurality of different crystallization samples within the one or
more lumens.
25. A method for determining crystallization conditions for a
material, the method comprising: taking a microfluidic device
comprising one or more lumens having microvolume dimensions and a
plurality of different crystallization samples within the one or
more lumens, the plurality of crystallization samples comprising a
material to be crystallized and crystallization conditions that
vary among the plurality of crystallization samples; transporting
the plurality of different crystallization samples within the one
or more lumens; identifying a precipitate or crystal formed in the
one or more lumens; and performing a spectroscopic analysis on the
identified precipitate or crystal while within the lumen.
26. A method according to claim 25, wherein the spectroscopic
analysis is selected from the group consisting of Raman, UV/VIS,
IR, and x-ray spectroscopy.
27. A method according to claim 25, wherein the spectroscopic
analysis is x-ray spectroscopy.
28. A method according to claim 27, wherein the x-ray spectroscopy
is x-ray diffraction.
29. A method according to claim 25, further including forming the
plurality of different crystallization samples within the one or
more lumens.
30. A method according to claim 25, wherein a plurality of
crystallization samples are comprised in at least one lumen.
31. A method according to claim 25, wherein transporting the
plurality of different crystallization samples within the one or
more lumens is performed by eletrokinetic. material transport.
32. A method according to claim 25, wherein transporting the
plurality of different crystallization samples within the one or
more lumens is performed by a method selected from the group
consisting of electrophoresis, electroosmotic flow and physical
pumping.
Description
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to microfluidic devices and
methods.
2. Description of Related Art
Traditional methods for crystal growth and crystallization are
highly labor intensive and require significant quantities of
material to evaluate and optimize crystal growth conditions.
Examples of these methods include the free interface diffusion
method (Salemme, F. R. (1972) Arch. Biochem. Biophys. 151:533-539),
vapor diffusion in the hanging or sitting drop method (McPherson,
A. (1982) Preparation and Analysis of Protein Crystals, John Wiley
and Son, New York, pp 82-127), and liquid dialysis (Bailey, K.
(1940) Nature 145:934-935).
Presently, the hanging drop method is the most commonly used method
for growing macromolecular crystals from solution, especially for
protein crystals. Generally, a droplet containing a protein
solution is spotted on a cover slip and suspended in a sealed
chamber that contains a reservoir with a higher concentration of
precipitating agent. Over time, the solution in the droplet
equilibrates with the reservoir by diffusing water vapor from the
droplet, thereby slowly increasing the concentration of the protein
and precipitating agent within the droplet, which in turn results
in precipitation or crystallization of the protein.
The process of growing crystals with high diffraction quality is
time-consuming and involves trial-and-error experiment on multiple
solution variables such as pH, temperature, ionic strength, and
specific concentrations of salts, organic additives, and
detergents. In addition, the amount of highly purified protein is
usually limited, multi-dimensional trials on these solution
conditions are unrealistic, labor-intensive and costly.
A few automated crystallization systems have been developed based
on the hanging drop methods, for example Cox, M. J. and Weber, P.
C. (1987) J. Appl. Cryst. 20:366; and Ward, K. B. et al. (1988) J.
Crystal Growth 90:325-339. More recently, systems for crystallizing
proteins in submicroliter drop volumes have been described
including those described in PCT Publication Nos. WO00/078445 and
WO00/060345.
Existing crystallization, such as hanging drop, sitting drop,
dialysis and other vapor diffusion methods have the limitation that
the material for analysis and the crystallization medium are
exposed to the environment for some time. As the volumes of
materials decrease, the ratio of surface area to volume ratio
varies as the inverse of the radius of the drop. This causes
smaller volumes to be more susceptible to evaporation during the
initial creation of the correct mixture and during the initial
period after the volume has been set up. Typical hanging drop
plates can have air volumes of 1.5 milliliters compared to a sample
drop size of 3-10 microliters. Moreover, typical methods expose the
sample drop to the environment for a duration of seconds to
minutes. Small variability in the rate that samples are made can
cause significant variations in the production of crystals. Small
variations external environment also can cause significant
variations in the production of crystals even if the rate that the
samples are made is unchanged. Prior methods fail to reduce the
problems of convection currents under 1 g such as those described
in U.S. Pat. No. 4,886,646, without the large expenditure of
resources or in methods that complicate crystal analysis.
SUMMARY OF THE INVENTION
The present invention relates to various microfluidics devices,
methods, and kits.
In one embodiment, a microfluidic device is provided that
comprises: a card shaped substrate having first and second opposing
faces; one or more microvolumes at least partially defined by a
first face of the card shaped substrate; and one or more grooves at
least partially defined by a second face of the card shaped
substrate; wherein a lateral footprint of at least a portion of the
one or more grooves overlaps with a lateral footprint of at least
one of the one or more microvolumes.
Optionally, the one or more grooves are sufficiently deep relative
to the second face of the substrate within the overlapping lateral
footprint that when the portion of the microvolume within the
overlapping lateral footprint comprises a crystallization sample
and an x-ray beam traverses the card shaped substrate at the
overlapping lateral footprint, the portion of the microvolume that
the x-ray beam traverses contains at least half as many electrons
as is contained in the substrate where the x-ray beam traverses.
Optionally, the portion of the microvolume that the x-ray beam
traverses contains at least as many electrons as is contained in
the substrate where the x-ray beam traverses. Preferably, the
portion of the microvolume that the x-ray beam traverses contains
at least three, five, ten times or more times as many electrons as
is contained in the substrate where the x-ray beam traverses.
Optionally, the one or more microvolumes comprise at least one
lumen. In such an instance, the groove may have a longitudinal axis
that is aligned with a longitudinal axis of the lumen adjacent the
overlapping lateral footprint. The groove may also have a
longitudinal axis that is perpendicular to a longitudinal axis of
the lumen adjacent the overlapping lateral footprint.
In another embodiment, a microfluidic device is provided that
comprises: a card shaped substrate having first and second opposing
faces; a plurality of microvolumes at least partially defined by a
first face of the card shaped substrate; and one or more grooves at
least partially defined by a second face of the card shaped
substrate; wherein a lateral footprint of at least a portion of the
one or more grooves overlaps with lateral footprints of plurality
of microvolumes.
In another embodiment, a method is provided for use with a
microfluidic device, the method comprising: performing an
experiment in a microfluidic device comprising a card shaped
substrate having first and second opposing faces, one or more
microvolumes at least partially defined by a first face of the card
shaped substrate; and one or more grooves at least partially
defined by a second face of the card shaped substrate; wherein a
lateral footprint of at least a portion of the one or more grooves
overlaps with a lateral footprint of at least one of the one or
more microvolumes; and performing a spectroscopic analysis within
the overlapping lateral footprint. Optionally, the microfluidic
device comprises a card shaped substrate.
In another embodiment, a method is provided for use with a
microfluidic device, the method comprising: performing an
experiment in a microvolume of a microfluidic device; and
performing a spectroscopic analysis using an x-ray beam that
traverses the microfluidic device such that material within the
microfluidic device that the x-ray beam traverses contains at least
as many electrons as is otherwise traversed when the x-ray beam
traverses the microfluidic device. Optionally, the material within
the microfluidic device that the x-ray beam traverses contains at
least three, five, ten times or more times as many electrons as is
otherwise traversed when the x-ray beam traverses the microfluidic
device.
In another embodiment, a method is provided for determining
crystallization conditions for a material, the method comprising:
taking a plurality of different crystallization samples in an
enclosed microvolume, the plurality of crystallization samples
comprising a material to be crystallized and crystallization
conditions which vary among the plurality of crystallization
samples; allowing crystals of the material to form in the plurality
of crystallization samples; and identifying which of the plurality
of crystallization samples comprise a precipitate, oil or a crystal
of the material. One or more dividers may optionally be positioned
between different crystallization samples in enclosed microvolume
to separate adjacent crystallization samples.
In another embodiment, a method is provided for determining
crystallization conditions for a material, the method comprising:
taking a plurality of different crystallization samples in a
plurality of enclosed microvolumes, each microvolume comprising one
or more crystallization samples, the crystallization samples
comprising a material to be crystallized and crystallization
conditions that vary among the plurality of crystallization
samples; allowing crystals of the material to form in plurality of
crystallization samples; and identifying which of the plurality of
crystallization samples comprise a precipitate, oil or a crystal of
the material. One or more dividers may optionally be positioned
between different crystallization samples in the enclosed
microvolumes to separate adjacent crystallization samples.
In another embodiment, a method is provided for determining
crystallization conditions for a material, the method comprising:
taking a microfluidic device comprising one or more lumens having
microvolume dimensions and a plurality of different crystallization
samples within the one or more lumens, the plurality of
crystallization samples comprising a material to be crystallized
and crystallization conditions-that vary among the plurality of
crystallization samples; transporting the plurality of different
crystallization samples within the lumens; and identifying a
precipitate or crystal formed in the one or more lumens.
Transporting the plurality of different crystallization samples
within the one or more lumens may be performed by a variety of
different methods. For example, transporting may be performed by a
method selected from the group consisting of electrophoresis,
electroosmotic flow and physical pumping. In one variation,
transporting is performed by electrokinetic material transport.
In a variation according to this embodiment, at least one of the
lumens optionally comprises a plurality of different
crystallization samples. One or more dividers may be positioned
between different crystallization samples in at least one of the
lumens to separate adjacent crystallization samples.
Also according to this embodiment, the method may further comprise
forming the plurality of different crystallization samples within
the one or more lumens. The plurality of crystallization samples
may be comprised in a single lumen or a plurality of lumens.
In another embodiment, a method is provided for determining
crystallization conditions for a material, the method comprising:
taking a microfluidic device comprising one or more lumens having
microvolume dimensions and a plurality of different crystallization
samples within the one or more lumens, the plurality of
crystallization samples comprising a material to be crystallized
and crystallization conditions that vary among the plurality of
crystallization samples; transporting the plurality of different
crystallization samples within the one or more lumens; and
identifying a precipitate or crystal formed in the one or more
lumens; and performing a spectroscopic analysis on the identified
precipitate or crystal while within the lumen.
The method may optionally further include forming the plurality of
different crystallization samples within the one or more lumens.
The plurality of crystallization samples may be comprised in a
single lumen or multiple lumens.
In another embodiment, a microfluidic method is provided
comprising: delivering a first fluid to a first lumen of a
microfluidic device and a second, different fluid to a second lumen
of the microfluidic device, the first and second lumens sharing a
common wall that allows for diffusion between the lumens over at
least a portion of the length of the lumens; and having the first
and second fluids diffuse between the first and second lumens.
In one variation according to this method, a composition of at
least one of the first and second fluids is varied so that the
composition of at least one of the first and second fluids varies
along a length of the lumen.
In another variation according to this method, the composition of
at least one of the first and second fluids varies over time as it
is delivered to the lumen so that the fluid forms a gradient with
regard to a concentration of at least one component of the fluid
that changes along a length of the lumen.
In another variation according to this method, the microfluidic
device comprises a plurality of first and second lumens, the method
comprising delivering first and second fluids to each of the
plurality of first and second lumens.
In yet another variation according to this method, the same first
and second fluids are delivered to each of the plurality of first
and second lumens.
In yet another variation according to this method, different first
and second fluids are delivered to the plurality of first and
second lumens.
It is noted that the first and second fluids may have a same or
different flow rate within the lumen. It is also noted that the
first and second fluids may each optionally comprise more than one
different fluid flow. The first and second fluids may also each
optionally comprise dividers that separate the fluid into a
plurality of aliquots separated by the dividers.
In another variation according to this method, the method
optionally further comprises delivering a third fluid to a third
lumen which shares a common wall with at least one of the first and
second lumens, the common wall allowing for diffusion between the
third lumen and the first or second lumen over at least a portion
of the length of the lumens.
In another embodiment, a microfluidic device is provided that
comprises: a substrate; a first lumen at least partially defined by
the substrate; and a second lumen; wherein the first and second
lumens share a common wall with each other that allows for
diffusion between the two lumens over at least a portion of the
length of the two lumens. The common wall may optionally comprise a
membrane, gel, frit, or matrix that allows for diffusion between
the two lumens.
Also according to this embodiment, the device may further comprise
a third lumen, the third lumen sharing a common wall with at least
one of the first and second lumens so as to allow for diffusion
between the lumens over at least a portion of the length of the
lumens.
In another embodiment, a microfluidic device is provided that
comprises: a substrate; a plurality of sets of lumens, each set
comprising a first lumen at least partially defined by the
substrate, and a second lumen, wherein the first and second lumens
share a common wall with each other that allows for diffusion
between the two lumens over at least a portion of the length of the
two lumens. The common wall may optionally comprise a membrane,
gel, frit, or matrix that allows for diffusion between the two
lumens.
According to this embodiment, the device may further comprise a
third lumen, the third lumen sharing a common wall with at least
one of the first and second lumens so as to allow for diffusion
between the lumens over at least a portion of the length of the
lumens.
Also according to this embodiment, the device may optionally
comprise at least 4, 8, 12, 24, 96, 200, 1000 or more sets of
lumens.
A variety of different devices and methods are also provided that
use centrifugal force to cause fluid movement within a microfluidic
device.
In one embodiment, a microfluidic method is provided that
comprises: taking a microfluidic device comprising a plurality of
microvolumes; and causing movement of material in a same manner
within the plurality of microvolumes by applying centrifugal forces
to the material.
In another embodiment, a microfluidic method is provided that
comprises: taking a plurality of microfluidic devices, each device
comprising a plurality of microvolumes; and causing movement of
material in a same manner within the plurality of microvolumes of
the plurality of devices by applying centrifugal forces to the
material. Optionally, a same centrifugal force is applied to each
of the plurality of devices.
In a variation, the plurality of microfluidic devices may be
stacked relative to each other when the centrifugal forces are
applied. The plurality of microfluidic devices may also be
positioned about a rotational axis about which the plurality of
microfluidic devices are rotated to apply the centrifugal
forces.
In another embodiment, a microfluidic method is provided that
comprises: taking a microfluidic device comprising a plurality of
microvolumes; and physically moving the device so as to effect a
same movement of material within the plurality of microvolumes.
Physically moving the device preferably causes centrifugal force to
be applied, for example, by rotation of the device about an
axis.
According to this embodiment, the material moved in each of the
plurality of microvolumes by movement of the device preferably has
a same quantity.
In another embodiment, a microfluidic method is provided that
comprises: taking a microfluidic device comprising a plurality of
microvolumes; and accelerating or decelerating a motion of the
device so as to effect a same movement of material within the
plurality of microvolumes. According to this embodiment, the motion
of the device is optionally a rotation of the device. In such
instances, acceleration or deceleration may be caused by a change
in a rate of rotation of the device.
In another embodiment, a microfluidic device is provided that
comprises: a substrate; and a plurality of microvolumes at least
partially defined by the substrate, each microvolume comprising a
first submicrovolume and a second submicrovolume that is in fluid
communication with the first submicrovolume when the device is
rotated, the plurality of microvolumes being arranged in the device
such that fluid in the first submicrovolumes of multiple of the
microvolumes are transported to second submicrovolumes of the
associated microvolumes when the device is rotated.
According to this embodiment, the device may be designed so that at
least 4, 8, 12, 36, 96, 200, 1000 or more of the microvolumes are
transported to second submicrovolumes of the associated
microvolumes when the device is rotated.
Also according to this embodiment, the device may be designed so
that the volume of fluid delivered from the first submicrovolume to
the second submicrovolume of a given microvolume upon rotation of
the device is within 50%, 25%, 10%, 5%, 2%, 1% or less of the
volume of fluid delivered from the first submicrovolumes to the
second submicrovolumes of any other microvolumes when a same volume
of fluid is added to the first submicrovolumes.
In another embodiment, a microfluidic method is provided that
comprises: taking a microfluidic device comprising a substrate, and
a plurality of microvolumes at least partially defined by the
substrate, each microvolume comprising a first submicrovolume and a
second submicrovolume where the first submicrovolume and second
microvolume are in fluid communication with each other when the
device is rotated; adding fluid to a plurality of the first
submicrovolumes; and rotating the device to cause fluid from the
plurality of first submicrovolumes to be transferred to the second
submicrovolumes in fluid communication with the first
submicrovolumes.
According to this embodiment, the device may be designed so that at
least 4, 8, 12, 24, 96, 200, 1000 or more of the microvolumes are
transported to second submicrovolumes of the associated
microvolumes when the device is rotated.
Also according to this embodiment, the device may be designed so
that the volume of fluid delivered from the first submicrovolume to
the second submicrovolume of a given microvolume upon rotation of
the device is within 50%, 25%, 10%, 5%, 2%, 1% or less of the
volume of fluid delivered from the first submicrovolumes to the
second submicrovolumes of any other microvolumes when a same volume
of fluid is added to the first submicrovolumes.
Also according to this embodiment, the method may be performed as
part of performing an array crystallization trial. The array
crystallization trial may involve the crystallization of a variety
of different materials including various biomolecules such as
proteins.
In another embodiment, a microfluidic method is provided that
comprises: taking a plurality of microfluidic devices, each
comprising a substrate, and a plurality of microvolumes at least
partially defined by the substrate, each sample microvolume
comprising a first submicrovolume and a second submicrovolume where
the first submicrovolume and second submicrovolume are in fluid
communication with each other when the device is rotated; adding
fluid to a plurality of the first submicrovolumes in the plurality
of microfluidic devices; and rotating the plurality of microfluidic
devices at the same time to cause fluid from the plurality of first
submicrovolumes to be transferred to the second submicrovolumes in
fluid communication with the first submicrovolumes.
According to this embodiment, the plurality of microfluidic devices
may optionally be stacked relative to each other during rotation.
The plurality of microfluidic devices may also be positioned about
a rotational axis about which the plurality of microfluidic devices
are rotated. In one variation, the rotational axis about which the
plurality of microfluidic devices are rotated is positioned within
the lateral footprints of the plurality of microfluidic devices. In
another variation, the rotational axis about which the plurality of
microfluidic devices are rotated is positioned outside of the
lateral footprints of the plurality of microfluidic devices.
In yet another embodiment, a microfluidic device is provided that
comprises: a substrate shaped so as to provide the device with an
axis of rotation about which the device may be rotated; and a
plurality of microvolumes at least partially defined by the
substrate, each microvolume comprising a first submicrovolume and a
second submicrovolume that is in fluid communication with the first
submicrovolume when the device is rotated, the plurality of
microvolumes being arranged in the device such that fluid in the
first submicrovolumes of multiple of the microvolumes are
transported to the second submicrovolumes of the associated
microvolumes when the device is rotated about the rotational axis.
Optionally, the second microvolumes are lumens.
The device may optionally comprise a mechanism that facilitates the
device being rotated about the rotational axis. For example, the
substrate may define a groove or hole at the rotational axis that
facilitates the device being rotated about the rotational axis.
Optionally, a center of mass of the device is at the rotational
axis and the substrate defines a groove or hole at the rotational
axis that facilitates the device being rotated about the rotational
axis. In one variation, the device is disc shaped, the substrate
defining a groove or hole at the rotational axis of the disc that
facilitates the device being rotated about the rotational axis.
Also according to this embodiment, the method may be performed as
part of performing an array crystallization trial. The array
crystallization trial may involve the crystallization of a variety
of different materials including various biomolecules such as
proteins.
In another embodiment, a microfluidic method is provided that
comprises: taking a microfluidic device comprising a substrate, and
a plurality of microvolumes at least partially defined by the
substrate, each microvolume comprising a first and a second
submicrovolume where the first and second submicrovolumes are in
fluid communication with each other when the device is rotated
about a rotational axis of the device; adding fluid to a plurality
of the first submicrovolumes; and rotating the device about the
rotational axis of the device to cause fluid in the first
submicrovolumes to be transferred to the second
submicrovolumes.
Also according to this embodiment, the method may be performed as
part of performing an array crystallization trial. The array
crystallization trial may involve the crystallization of a variety
of different materials including various biomolecules such as
proteins.
In another embodiment, a microfluidic device is provided that
comprises: a substrate; one or more microvolumes at least partially
defined by the substrate, each microvolume comprising a first
submicrovolume, a second submicrovolume where fluid in the first
submicrovolume is transported to the second submicrovolume when the
device is rotated about a first rotational axis, and a third
submicrovolume where fluid in the first submicrovolume is
transported to the third submicrovolume when the device is rotated
about a second, different rotational axis. The device itself
include features to facilitate the rotation of the device about one
or more rotational axes. The device may alternative be rotated
about one or more rotational axes by the use of an external
fixture.
In another embodiment, a microfluidic device comprising: a
substrate; one or more microvolumes extending along a plane of the
substrate, each microvolume comprising a first submicrovolume, a
second submicrovolume where fluid in the first submicrovolume is
transported to the second submicrovolume when the device is rotated
about a first rotational axis that is positioned further away from
the second submicrovolume than the first submicrovolume, and a
third submicrovolume where fluid in the first submicrovolume is
transported to the third submicrovolume when the device is rotated
about a second, different rotational axis that is positioned
further away from the third submicrovolume than the first
submicrovolume. Optionally, the substrate is card shaped. In such
instances, the one or more microvolumes may optionally extend along
a surface of the card shaped substrate.
In another embodiment, a microfluidic method is provided that
comprises: taking a microfluidic device comprising a substrate and
a plurality of microvolumes at least partially defined by the
substrate, each microvolume comprising an first submicrovolume, a
second submicrovolume where fluid in the first submicrovolume is
transported to the second submicrovolume when the device is rotated
about a first rotational axis, and a third submicrovolume where
fluid in the first submicrovolume is transported to the third
submicrovolume when the device is rotated about a second, different
rotational axis; adding fluid to the first submicrovolumes of the
microvolumes; and in any order rotating the device about the first
and second rotational axes to cause fluid from the first
submicrovolumes to be transferred to the second and third
submicrovolumes.
It is noted that the method may be performed as part of performing
an array crystallization trial. The array crystallization trial may
involve the crystallization of a variety of different materials
including various biomolecules such as proteins.
In another embodiment, a microfluidic device is provided that
comprises: a substrate; and a plurality of microvolumes at least
partially defined by the substrate, each microvolume comprising a
first submicrovolume and a second submicrovolume in fluid
communication with the first submicrovolume when the device is
rotated about a first rotational axis, wherein rotation of the
device about the first rotational axis causes a fixed volume to be
transported to each of the second submicrovolumes.
According to this embodiment, the plurality of microvolumes may
optionally further comprise one or more outlet submicrovolumes in
fluid communication with the first submicrovolume.
Also according to this embodiment, the plurality of microvolumes
may optionally further comprise one or more outlet submicrovolumes
where fluid in the first submicrovolume not transported to the
second submicrovolume when the device is rotated about a first
rotational axis is transported to one or more one or more outlet
submicrovolumes when the device is rotated about a second,
different rotational axis.
In another embodiment, a microfluidic device is provided that
comprises: a substrate; a first microvolume at least partially
defined by the substrate comprising a first submicrovolume; a
second submicrovolume where fluid in the first submicrovolume is
transported to the second submicrovolume when the device is rotated
about a first rotational axis; and a second microvolume at least
partially defined by the substrate comprising a third
submicrovolume; a fourth submicrovolume where fluid in the third
submicrovolume is transported to the fourth submicrovolume when the
device is rotated about the first rotational axis; and wherein
fluid in the second and fourth submicrovolumes are transported to a
fifth submicrovolume where the second and fourth submicrovolumes
are mixed when the device is rotated about a second, different
rotational axis.
According to this embodiment, the fifth submicrovolume may
optionally be in fluid communication with the second and fourth
submicrovolumes via the first and third submicrovolumes
respectively.
Also according to this embodiment, the device may further comprise
one or more outlet submicrovolumes in fluid communication with the
first and third submicrovolumes.
Also according to this embodiment, the device may further comprise
one or more outlet submicrovolumes in fluid communication with the
first and second submicrovolumes where fluid in the first and third
submicrovolumes not transported to the second and fourth
submicrovolumes when the device is rotated about the first
rotational axis is transported to one or more one or more outlet
submicrovolumes when the device is rotated about a third, different
rotational axis.
Also according to this embodiment, the device may further comprise
at least 4, 8, 12, 24, 96, 200, 1000, or more pairs of first and
second microvolumes.
Also according to this embodiment, the device may be designed such
that the volume of fluid transported to any given second
submicrovolume does not deviate from the volume of fluid
transported to another second submicrovolume by more than 50%, 25%,
10%, 5%, 2%, 1% or less.
The device may also optionally be designed so that any of the
following conditions are satisfied: the first rotational axis is
positioned further away from the second and fourth submicrovolumes
than the first and third submicrovolumes; the first rotational axis
about which the microfluidic device is designed to be rotated is
positioned within a lateral footprint of the microfluidic device;
and the first rotational axis about which the microfluidic device
is designed to be rotated is positioned outside of a lateral
footprint of the microfluidic device.
In yet another embodiment, a microfluidic method is provided that
comprises: taking a microfluidic device comprising a substrate, and
a plurality of microvolumes at least partially defined by the
substrate, each microvolume comprising a first submicrovolume and a
second submicrovolume in fluid communication with the first
submicrovolume; adding fluids to the first submicrovolumes; and
applying a centrifugal force to the device to cause a same volume
of fluid to be transported to the second microvolumes from the
first submicrovolumes.
Optionally, the microvolumes may further comprise an outlet
submicrovolume in fluid communication with the first
submicrovolumes. In such instances, the method may further comprise
transporting fluid in the first submicrovolume to the outlet
submicrovolume that was not transported to the second
submicrovolume when the centrifugal force was applied. The method
may also further comprise transporting fluid in the first
submicrovolume to the outlet submicrovolume that was not
transported to the second submicrovolume when the device is rotated
about a first rotational axis by rotating the device about a
second, different rotational axis.
Also according to the embodiment, the device may be designed such
that the volume of fluid transported to any given second
submicrovolume does not deviate from the volume of fluid
transported to another second submicrovolume by more than 50%, 25%,
10%, 5%, 2%, 1% or less.
In another embodiment, a microfluidic method is provided that
comprises: taking a microfluidic device comprising a substrate, a
first microvolume at least partially defined by the substrate
comprising a first submicrovolume and a second submicrovolume where
fluid in the first submicrovolume is transported to the second
submicrovolume when the device is rotated about a first rotational
axis, and a second microvolume at least partially defined by the
substrate comprising a third submicrovolume and a fourth
submicrovolume where fluid in the third submicrovolume is
transported to the fourth submicrovolume when the device is rotated
about the first rotational axis, the microvolumes further
comprising a fifth submicrovolume where fluid in the second and
fourth submicrovolumes are mixed when the device is rotated about a
second, different rotational axis; adding a first fluid to the
first submicrovolume and a second fluid to the third
submicrovolume; rotating the device about the first rotational axis
to transport the first and second fluids to the second and fourth
submicrovolumes; and rotating the device about the second
rotational axis to transport the first and second fluids from the
second and fourth submicrovolumes to the fifth submicrovolume.
In one variation, the fifth submicrovolume is in fluid
communication with the second and fourth submicrovolumes via the
first and third submicrovolumes respectively.
Optionally, the method further comprises removing fluid from the
first and third submicrovolumes that is not transported to the
second and fourth submicrovolumes prior to rotating the device
about the second rotational axis.
Also according to the embodiment, the device may comprise a
plurality of pairs of first and second microvolumes and the volume
of fluid transported to any given second submicrovolume does not
deviate from the volume of fluid transported to another second
submicrovolume by more than 50%, 25%, 10%, 5%, 2%, 1% or less.
In another embodiment, a microfluidic method is provided that
comprises: delivering first and second fluids to a lumen of a
microfluidic device such that the first and second fluids flow
adjacent to each other within the lumen without mixing except for
diffusion at an interface between the first and second fluids,
wherein the first fluid is different than the second fluid.
According to this embodiment, the composition of at least one of
the first and second fluids is optionally varied over time as it is
delivered to the lumen so that the fluid forms a gradient with
regard to a concentration of at least one component of the fluid
that changes along a length of the lumen.
According to this embodiment, the microfluidic device may comprise
a plurality of lumens, the method optionally comprising delivering
first and second fluids to each of the plurality of lumens.
According to this embodiment, the same first and second fluids may
be delivered to each of the plurality of lumens. Alternatively,
different first and second fluids are delivered to the different
lumens of the plurality of lumens. The first and second fluids may
also have a same or different flow rate within the lumen.
Also according to this embodiment, the first and second fluids may
be combined to form different crystallization conditions for
crystallizing a molecule such as a protein.
In another embodiment, a microfluidic method is provided that
comprises: delivering first and second fluids to a lumen of a
microfluidic device such that the first and second fluids flow
adjacent to each other within the lumen without mixing except for
diffusion at an interface between the first and second fluids,
wherein the first fluid is different than the second fluid and a
composition of at least one of the first and second fluids
delivered to the lumen is varied so that the composition of at
least one of the first and second fluids within the lumen varies
along a length of the lumen.
In yet another embodiment, a microfluidic method is provided that
comprises: delivering first, second and third fluids to a lumen of
a microfluidic device such that the first, second and third fluids
flow adjacent to each other within the lumen without mixing except
for diffusion at an interface between the first, second and third
fluids, wherein the first, second and third fluids are different
than each other and a composition of at least one of the first,
second and third fluids delivered to the lumen is varied so that
the composition of at least one of the first, second, and third
fluids within the lumen varies along a length of the lumen.
According to this embodiment, the composition of at least one of
the first, second and third fluids may be varied over time as it is
delivered to the lumen so that the fluid forms a gradient with
regard to a concentration of at least one component of the fluid
that changes along a length of the lumen.
Also according to this embodiment, the microfluidic device may
comprise a plurality of lumens, the method comprising delivering
first, second and third fluids to each of the plurality of
lumens.
The same or different first, second and third fluids may be
delivered to each of the plurality of lumens. Optionally, at least
one of the first, second and third fluids have a different flow
rate than another of the fluids within the lumen. Also, at least
one of the first, second and third fluids may have the same flow
rate than another of the fluids within the lumen.
Also according to this embodiment, the first, second and third
fluids may be combined to form different crystallization conditions
for crystallizing a molecule such as a protein. In one variation,
the first, second and third fluids combine to form different
crystallization conditions, the second fluid comprising the
material to be crystallized and being positioned between the first
and third fluids.
In regard to the various embodiments where a device is rotated
about one or more rotational axes, the device may optionally be
designed so that any or more of the following conditions are
satisfied: the first and second rotational axes are laterally
offset relative to each other; the first and second rotational axes
are at an angle relative to each other and intersect; the first and
second rotational axes are at an angle relative to each other and
are laterally offset; the first and second rotational axes are
perpendicular to each other and intersect; the first and second
rotational axes are perpendicular to each other and are laterally
offset; the first and second rotational axes are at an angle of 45
degrees relative to each other and intersect; the first and second
rotational axes are at an angle of 45 degrees relative to each
other and are laterally offset; and the first and second rotational
axes are parallel and laterally offset relative to each other.
According to any of the embodiments employing centrifugal forces,
the devices may be designed so that material is optionally moved
within at least 4, 8, 12, 24, 96, 200, 1000 or more different
microvolumes in a same manner when the centrifugal forces are
applied.
Also according to any of the embodiments employing centrifugal
forces, the devices may be designed so that the volume of fluid or
other material delivered to a submicrovolume in a given microvolume
is within 50%, 25%, 10%, 5%, 2%, 1% or less of the volume of fluid
or other material delivered to a corresponding submicrovolume in
any other microvolume.
Optionally, the centrifugal forces are applied such that a same
centrifugal force is applied to material in each of the plurality
of microvolumes.
Optionally, the centrifugal forces are applied such that at least
0.01 g, 0.1, 1 g, 10 g, 100 g or more force is applied to the
material in the device to cause the material to move within the
microvolumes.
Applying the centrifugal forces may be performed by rotating the
device. Optionally, the centrifugal forces are applied by rotating
the device at least 10 rpm, 50 rpm, 100 rpm or more.
In regard to all of the above embodiments, unless otherwise
specified, microvolumes may have a variety of shapes including, but
not limited to lumens and microchambers. When a lumen is employed,
the lumen optionally has a cross sectional diameter of less than
2.5 mm, optionally less than 1 mm, and optionally less than 500
microns.
A variety of different substrates may be used to make the
microfluidic devices of the present invention. In one variation,
the substrate comprises one or more members of the group consisting
of polymethylmethacrylate, polycarbonate, polyethylene
terepthalate, polystyrene, styrene copolymers, glass, and fused
silica. In one variation, the substrate is optically
transparent.
According to each of the above embodiments, the experiment being
performed may optionally be a crystallization of a molecule or
material. The crystallization may optionally be of a biomolecule.
Examples of biomolecules that may be crystallized include, but are
not limited to viruses, proteins, peptides, nucleosides,
nucleotides, ribonucleic acids, and deoxyribonucleic acids.
It is also noted that the material to be crystallized may contain
one, two or more materials selected from the group consisting of
viruses, proteins, peptides, nucleosides, nucleotides, ribonucleic
acids, deoxyribonucleic acids, small molecules, drugs, putative
drugs, inorganic compounds, metal salts, organometallic compounds
and elements. In one variation, the material to be crystallized is
a macromolecule with a molecular weight of at least 500
Daltons.
In certain embodiments, a spectroscopic analysis is performed. The
spectroscopic analysis may optionally be-selected from the group
consisting of Raman, UV/VIS, IR, x-ray spectroscopy, polarization,
and fluorescent. In one particular variation, the spectroscopic
analysis is x-ray spectroscopy. In a further particular variation,
the x-ray spectroscopy is x-ray diffraction.
In some instances, the spectroscopic analysis involves an x-ray
traversing the microfluidic device. In such instances, a groove may
be employed in the device that is sufficiently deep relative to the
second face of the substrate within the overlapping lateral
footprint that when the portion of the microvolume within the
overlapping lateral footprint comprises a crystallization sample
and an x-ray beam traverses the card shaped substrate at the
overlapping lateral footprint, the portion of the microvolume that
the x-ray beam traverses contains at least half as many electrons
as is contained in the substrate where the x-ray beam traverses.
Optionally, the portion of the microvolume that the x-ray beam
traverses contains at least as many electrons as is contained in
the substrate where the x-ray beam traverses. Preferably, the
portion of the microvolume that the x-ray beam traverses contains
at least three, five, ten times or more times as many electrons as
is contained in the substrate where the x-ray beam traverses.
Each of the above embodiments may optionally include transporting
material within the microfluid device. Such transport may be
performed by a variety of different methods. For example,
transporting may be performed by a method selected from the group
consisting of electrophoresis, electroosmotic flow and physical
pumping. In one variation, transporting is performed by
electrokinetic material transport. In some instances, transporting
is performed by moving the device. This may be done by applying a
centrifugal force, which in turn may be performed by rotating the
device about a rotational axis.
Each of the above embodiments may optionally include the use of one
or more dividers to separate aliquots of materials. In some
instances, the separated aliquots of materials correspond to
separate experiments such as crystallization trials. The dividers
may be formed of a variety of different materials. For example, the
dividers may be formed of a permeable, semi-permeable or
impermeable material that may be a gas, liquid, gel, or solid. In
one particular variation, the one or more dividers are selected
from the group consisting of a membrane, gel, frit, and matrix.
The one or more dividers may form various interfaces including
those selected from the group consisting of liquid/liquid,
liquid/gas interface, liquid/solid and liquid/sol-gel
interface.
The one or more dividers optionally function to modulate diffusion
characteristics between adjacent crystallization samples. For
example, the one or more dividers may be formed of a semi-permeable
material that allows diffusion between adjacent crystallization
samples.
These and other methods, devices, compositions and kits are
described herein.
BRIEF DESCRIPTION OF FIGURES
FIG. 1A illustrates a card shaped device housing microvolumes with
opposing faces.
FIG. 1B illustrates an embodiment of a card shaped device where the
thickness of the overall card is reduced adjacent a region where
x-rays will be incident in order to reduce the amount of material
in the path of the x-rays.
FIG. 1C illustrates a bottom up view of an embodiment of a card
shaped device where grooves have been created so that less material
is present adjacent a region where x-rays will be incident.
FIG. 1D illustrates a cross sectional view of an embodiment of a
card shaped device where grooves have been created so that less
material is present adjacent a region where x-rays will be
incident.
FIG. 2 illustrates the generalized use of a microvolume dimensioned
lumen to form crystallization samples and perform
crystallization.
FIG. 3A illustrates various interconnections that may be formed
between different lumens.
FIG. 3B illustrates how two sub-lumens extending from and joining
with a main lumen may be used to effect mixing within the main
lumen.
FIG. 3C illustrates the use of a dividing feature to separate a
crystal containing crystallization experiment into two
portions.
FIG. 3D illustrates different combinations of single and double
ports that may be combined for complex mixing, separation,
diffusion and purifications.
FIGS. 4A-4C provide several embodiments of performing
crystallizations within a lumen.
FIG. 4A illustrates a crystallization mixture performed within a
lumen positioned between two dividers.
FIG. 4B illustrates a crystallization performed within a lumen
where multiple crystallization conditions are simultaneously
employed.
FIG. 4C illustrates a crystallization performed within a lumen
where a series of crystallization agents are set up for
crystallization against a series of substances to be
crystallized.
FIG. 5A illustrates a crystallization performed within a lumen
where one or more of the elements of the crystallization experiment
change along a length of the lumen. The change can occur discretely
or continuously, and need not be changed in a simple linear
method.
FIG. 5B illustrates a crystallization performed within a lumen
where a series of substances to be crystallized are in a single
gradient.
FIG. 5C illustrates a crystallization performed within a lumen
where a series of crystallization agents can be assayed against a
substance to be crystallized.
FIG. 5D illustrates diffusion between various elements in a
crystallization performed within a lumen.
FIG. 6A illustrates a crystallization performed within a lumen
where a single crystallization condition occupies an entire
crystallization space.
FIG. 6B illustrates multiple crystallizations being performed
within a lumen where dividers are used between the
crystallizations, the dividers being shown to have planar surfaces
adjacent the crystallizations.
FIG. 6C illustrates multiple crystallizations being performed
within a lumen where dividers are used between the
crystallizations, the dividers being shown to have curved, convex
surfaces adjacent the crystallizations.
FIG. 7A shows a device for performing a series of crystallizations
within a series of lumens where each lumen comprises a loading and
unloading port and a lumen body interconnecting the ports.
FIG. 7B shows a cross section of a device for performing a
crystallization within a lumen where the lumen is not enclosed.
FIG. 7C shows a cross section of a device for performing a
crystallization within a lumen where the lumens are rectangular in
shape.
FIG. 7D shows a cross section of a device for performing a
crystallization within a lumen where the lumens are curved or
tubular in shape.
FIG. 7E shows a device for performing crystallizations within a
series of lumens where the lumens are loaded with samples that are
separated by divider or modifier segments. It should be appreciated
that each discrete sample may have conditions that are potentially
unique and unrelated to adjacent samples. The dividers or modifiers
positioned between the samples can be permeable, semi-permeable or
impermeable.
FIG. 8A shows a device for performing a series of different
crystallizations within a series of lumens where each lumen
comprises a loading and unloading port and a lumen body
interconnecting the ports.
FIG. 8B illustrates a single lumen in which a barrier is adjacent
to the crystallization condition bounded by second barrier.
FIG. 8C illustrates a lumen comprising a more complex design of
crystallizations than the lumen shown in FIG. 8B.
FIG. 8D illustrates a multi-component crystallization being
performed in a single lumen.
FIG. 9A shows an embodiment of a device for performing a series of
different crystallizations within a series of lumens where each
lumen comprises a loading and unloading port and a lumen body
interconnecting the ports.
FIG. 9B illustrates an enlargement of a lumen of the device shown
in FIG. 9A which illustrates some of the different simultaneous
diffusions that are made possible by the invention.
FIG. 9C illustrates the device shown in FIG. 9B where diffusion
occurred through the barrier to form a gradient from condition to
condition.
FIG. 9D illustrates crystals forming at different locations after
the diffusion shown in FIG. 9C.
FIG. 10A illustrates an embodiment of a device comprising first and
second lumens where the first and second lumens share a common wall
that allows for diffusion between the lumens over at least a
portion of the length of the lumens.
FIG. 10B illustrates an embodiment of a device comprising first,
second and third lumens where the first, second and third lumens
share a common wall that allows for diffusion between the lumens
over at least a portion of the length of the lumens.
FIG. 10C illustrates a crystallization experiment loaded into a
double lumen device such as the device shown in FIG. 10A.
FIG. 10D illustrates diffusion having occurred both through the
semi-permeable internal divider, as well as through the permeable
or semi-permeable wall of the device shown in FIG. 10C.
FIG. 10E illustrates the experiment shown in FIG. 10D where a
series of crystal growths have occurred after some diffusion has
occurred.
FIG. 10F illustrates a device with multiple lumens that may each be
separated by permeable or semi-permeable wall.
FIG. 11A illustrates a single lumen with integral mixing and
harvesting channels.
FIG. 11B shows an embodiment of a device for performing a series of
different crystallizations within a series of lumens where each
lumen comprises integral mixing and harvesting channels.
FIG. 12A illustrates a device comprising a series of lumens, each
lumen having attached to it an array of individual crystallization
cells, each cell having at least one separate inlet or outlet and
at least one channel connecting the cell to the lumen.
FIG. 12B illustrates an embodiment of an individual crystallization
cell shown in FIG. 12A.
FIG. 12C illustrates an embodiment of an individual crystallization
cell shown in FIG. 12A where the cell comprises a crystallization
agent and a substance to be crystallized.
FIG. 13 illustrates a device for forming crystallizations by
rotation of the device.
FIG. 14 illustrates a device that is designed to move fluids within
the device by centrifugal force.
FIGS. 15A-15G illustrate an embodiment of a centrifugally driven
crystallization device.
FIG. 15A illustrates a repeating unit of the centrifugal array.
FIG. 15B illustrates a process for using a centrifugal device.
FIG. 15C illustrates the effect of centrifugal force on the samples
that are loaded in the centrifugal device illustrated in FIG.
15B.
FIG. 15D illustrates what happens when the centrifugal force vector
is changed such that the force now directs the excess
crystallization agent and excess material to be crystallized toward
the waste ports via the respective waste channels.
FIG. 15E illustrates each channel of the centrifugal device filled
to point V, resulting in precise volume measurements.
FIG. 15F illustrates what happens when the centrifugal force vector
has been altered to align in the direction shown.
FIG. 15G illustrates crystallization chamber filled with the
combination of the material to be crystallized and the
crystallization agent, or agents.
DETAILED DESCRIPTION OF THE INVENTION
The present invention relates to various methods, devices and kits
relating to microfluidics.
One particular aspect of the present invention relates to the use
of these methods and devices for forming crystallization samples,
transporting crystallization samples, and crystallizing materials
therein, particularly on a microvolume scale, high throughput
manner. Distinguishing the present invention in this regard is the
performance of the crystallizations in very small, substantially
enclosed volumes formed by or within a substrate, referred to
herein as an "enclosed microvolume". Other aspects of the present
invention will be understood by one of ordinary skill in view of
the teachings provided herein.
It is noted that many of the particular embodiments are described
herein in regard to performing crystallization experiments.
However, it should be understood that many of the operations
involved in performing crystallization experiments (e.g.,
measuring, mixing, fluid flow and analysis) made possible by the
various devices and methods of the present invention have
applications outside of performing crystallization experiments and
should therefore not be limited to crystallization experiments.
A "crystallization sample", as the term is used herein, refers to a
mixture comprising a material to be crystallized. The
crystallization includes such other components in the mixture to
cause or at least attempt to cause crystals of the material to be
formed in the mixture.
According to the present invention, crystallization samples are
formed, transported, and crystallization attempts conducted in
enclosed microvolumes. These enclosed microvolumes comprise one or
more lumens and optionally microchambers in fluid communication
with the lumens. The lumens are enclosed within a substrate. When
employed, microchambers are enclosed microvolumes defined within
the substrate in fluid communication with the lumens. The lumens
and microchambers provide an encased environment within which
crystallization samples may be formed, and crystallization attempts
performed and analyzed.
The term "lumen" as the term is used herein, refers to any
elongated, enclosed volume formed at least partially by a
substrate. The lumen preferably has a cross sectional diameter of
less than 2.5 mm, preferably less than 1 mm and more preferably
less than 500 microns. In one variation, the lumen has a cross
sectional diameter between 0.1 microns and 2.5 mm, preferably
between 0.1 microns and 1 mm, and preferably between 0.1 and 500
microns. Aside from openings in the lumen, most typically adjacent
the proximal and distal ends of the lumen, the lumen provides an
enclosed environment in which to form, transport, conduct, and
optionally analyze crystallizations.
Mass flow may be reduced by controlling the length of the
crystallization volume within the microlumens. This serves to
reduce the forces driving convection currents within the
crystallization condition. By minimizing the length of the
crystallization volume within the microlumens, facile control of
the degree of convection currents within the microlumen is
controlled.
In certain instances, it may be desirable for the lumen to be in
fluid communication with one or more microchambers. A
"microchamber", as the term is used herein, refers to a volume in
fluid communication with a lumen that has a larger cross sectional
area than the lumen.
By forming crystallization conditions and performing
crystallizations within the small, relatively sealed volumes
defined by the enclosed microvolumes of the lumens and
microchambers, a variety of different advantages are provided.
One advantage provided by conducting crystallizations in enclosed
microvolumes is that it facilitates parallel screening of many
materials at once or a material in many conditions at once, or a
combination thereof.
A further advantage provided by the small volumes associated with
performing crystallizations in enclosed microvolumes is that it
enables the conservation of the material to be crystallized,
thereby enabling greater numbers of crystallization conditions to
be sampled using a given amount of material. By achieving higher
densities of crystallization conditions, advancements in crystal
analysis are obtained.
A further advantage provided by performing crystallizations
according to the present invention is a reduction in evaporation
during the preparation and performance of the crystallization. As a
result, crystallization conditions can be more precisely controlled
and remain stable for longer periods of time. Crystallizations can
also be conducted over a wider range of temperature conditions
since losses due to evaporation are significantly curtailed.
A further advantage provided by performing crystallizations
according to the present invention is a further reduction in the
space requirements for performing crystallizations. More
specifically, the present invention allows multiple
crystallizations to be performed in a denser format. This allows
the device within which the crystallizations are performed to be
smaller and allows more crystallizations to be performed in a
single device. For example, when in situ crystallizations are
performed in a thin cassette or card, the crystallizations may be
densely packed, allowing for rapid and efficient analysis of the
crystallization conditions.
A further advantage provided by performing crystallizations
according to the present invention is the more rapid equilibration
times that may be achieved by further reducing crystallization
volumes.
A further advantage provided by performing crystallizations
according to the present invention is the number of parallel
experiments that may be performed. For example, embodiments of the
present invention provide for the use of at least 4, 8, 12, 24, 96,
200, 1000 or more different microvolumes in parallel. In regard to
the use of centrifugal forces, the devices may be designed so that
material is moved within at least 4, 8, 12, 24, 96, 200, 1000 or
more different microvolumes in a same manner when the centrifugal
forces are applied.
Yet a further advantage provided by performing crystallizations
according to the present invention is the precision with which
fluid and material can be transported. For example, certain
embodiments use centrifugal forces to transport materials. By being
able to closely control the sizes of the microvolumes, devices can
be designed so that the volume of fluid delivered to a given
submicrovolume of a given microvolume upon rotation of the device
is within 50%, 25%, 10%, 5%, 2%, 1% or less of the volume of fluid
delivered to submicrovolumes of other microvolumes.
As will be evident from the foregoing description of the operation
of the devices of the present invention, a further advantage
provided is simplified material handling.
1. Materials To Be Crystallized
While problems associated with crystal growth addressed by the
present invention are of particular interest for proteins and other
biomolecules, it is a general problem of all crystal forming
materials. The materials to be crystallized may be any substance
capable of crystallizing or co-crystallizing. For example, the
material to be crystallized may contain one, two or more materials
selected from the group consisting of viruses, proteins, peptides,
nucleosides, nucleotides, ribonucleic acids, deoxyribonucleic
acids, ligands, small molecules, drugs, putative drugs, inorganic
compounds, metal salts, organometallic compounds and elements and
mixtures and combinations thereof.
The materials to be crystallized may be any material for which a
crystal structure is needed. Determining high-resolution structures
of materials by a high-throughput method such as the one of the
present invention can be used to accelerate the analysis of
materials, especially drug development.
The material to be crystallized may also be a molecule for which a
crystalline form of the molecule is needed. For example, it may be
desirable to create a crystalline form of a molecule or to identify
new crystalline forms of a molecule. In some instances, particular
crystalline forms of a molecule may have greater biological
activity, dissolve faster, decompose less readily, and/or be easier
to purify.
The material to be crystallized may also be a-combination of
substances for the production of co-crystals. The co-crystals can
comprise any two of a small molecule, a drug, a ligand, a
substrate, an inhibitor, a guest chemical, protein, nucleotide, or
a protomer. The substances can be a plurality of small molecules,
drugs, ligands, substrates, inhibitors, guest chemicals, proteins,
or a protomers.
The material to be crystallized is preferably a macromolecule such
as a protein but may also be other types of macromolecules. The
molecule preferably has a molecular weight of at least 500 Daltons,
more preferably at least 1000 Daltons, although smaller molecular
weight molecules may also be crystallized.
2. Construction of Enclosed Microvolumes
The construction, design and operation of various different
microfluidic devices have been described in literature and are thus
known in the art. For example, U.S. Pat. Nos. 5,126,022; 5,296,114;
5,180,480; 5,132,012; and 4,908,112 are examples of references
detailing the design and construction of lumens and microchambers
in a substrate. Other examples of references include Harrison et
al., "Micromachining a Miniaturized Capillary Electrophoresis-Based
Chemical Analysis System on a Chip," Science (1992) 261: 895;
Jacobsen et al., "Precolumn Reactions with Electrophoretic Analysis
Integrated on a Microchip," Anal. Chem. (1994) 66: 2949;
Effenhauser et al., "High-Speed Separation of Antisense
Oligonucleotides on a Micromachined Capillary Electrophoresis
Device," Anal. Chem. (1994) 66:2949; and Woolley & Mathies,
"Ultra-High-Speed DNA Fragment Separations Using Capillary Array
Electrophoresis Chips," P.N.A.S. USA (1994) 91:11348. Further
examples of different microfluidic devices include, but are not
limited to those described in: U.S. Pat. Nos. 6,306,273, 6,284,113,
6,176,962, 6,103,537, 6,093,296, 6,074,827, 6,007,690, 5,858,188,
5,126,022, 5,750,015, 5,935,401, 5,770,029 assigned to Aclara,
Inc.; U.S. Pat. Nos. 6,321,791, 6,316,781, 6,316,201, 6,306,272,
6,274,337, 6,274,089, 6,267,858, 6,251,343, 6,238,538, 6,235,175,
6,221,226, and 6,186,660 assigned to Caliper, Inc.; PCT Application
Nos. WO 01/94635, WO 01/75176, WO 01/67369, WO 01/32930, WO
01/01025, WO 99/61888, each assigned to Fluidigm, Inc.; U.S. Pat.
Nos. 6,319,472, 6,238,624 assigned to Nanogen, Inc.; U.S. Pat. Nos.
6,290,685 assigned to 3M Corp.; as well as U.S. Pat. Nos.
6,261,430, 6,251,247, 6,236,945, 6,210,986, 6,176,990, 6,007,690,
6,074,827, 6,056,860, 6,054,034, 5,885,470, 5,858,195, 5,750,015,
5,571,410, 5,580,523, 5,296,114, 5,180,480, 5,132,012, 5,126,022,
4,891,120, and 4,908,112.
It should be understood that these numerous examples are only
intended to be illustrative in regard how enclosed microvolumes
according to the present invention may be constructed, designed and
operated in conjunction with the present invention.
Transport of material within the microfluidic devices of the
present invention may be performed by any mode of transport
available to microfluidic devices including, but not limited to
electrophoresis, electroosmotic flow and physical pumping. In one
variation, transporting is performed by electrokinetic material
transport. A novel feature of certain embodiments of the present
invention, discussed herein in greater detail, is the use of
centrifugal force to transport material, for example by rotating
the device about a rotational axis.
The enclosed microvolumes may be formed in any substrate within
which microvolumes may be formed. Examples of suitable substrates
include, but are not limited to glass, fused silica, acrylics,
thermoplastics, and the like. The various components of the
integrated device may be fabricated from the same or different
materials, depending on the particular use of the device, the
economic concerns, solvent compatibility, optical clarity, color,
mechanical strength, and the like.
For applications where it is desired to have a disposable device,
due to ease of manufacture and cost of materials, the device will
typically be fabricated from a plastic. For ease of detection and
fabrication, the entire device may be fabricated from a plastic
material that is optically transparent, as that term is defined
above. Particular plastics finding use include
polymethylmethacrylate, polycarbonate, polyethylene terepthalate,
polystyrene or styrene copolymers, and the like. It is noted that
these various materials may be used alone or in combination to form
the devices of the present invention.
The substrate comprising the enclosed microvolumes may be in any
form, e.g., a tube, a card, a chip or a block. The substrate is
preferably in the form of a card. The card preferably has a face
sized less than 12 cm.times.8.5 cm.
The enclosed microvolumes may be formed by any process by which an
enclosed lumen or chamber may be created in a material. For
example, the shape of the substrate and the enclosed microvolumes
may be formed by thermoplastic injection molding, micromolding,
punching, milling, any solid free form technology, such as three
dimensional printing, or other types of manufacturing technologies
for plastics, such as micromolding, embossing, laser drilling,
extrusion, injection, or electron deposition machining, glass or
silicon, conventional silicon processing technology, such as
photolithography, deep reactive ion or wet etching, electron beam
machining, micromachining, electro-discharge machining, reaction
injection molding.
It is noted that the substrate comprising the enclosed microvolume
may be formed of a single material, such as a block or a card.
Alternatively, one or more materials may be brought together to
form the enclosed microvolume. This typically involves having a
portion of the microvolume be formed by a first substrate (e.g.,
photolithography on a surface of the first substrate). A second
substrate is brought together with the first substrate to complete
the definition of the enclosed microvolume. The act of combining
the first and second substrates can cause the material to be
crystallized to be enclosed. The act of combining can also cause
mixing to occur. The substrate is preferably optically clear,
transparent, translucent or opaque.
The substrate is preferably formed of a material that allows for
various spectroscopic analyses (e.g., Raman, UV/VIS, IR or x-ray
spectroscopy, polarization, fluorescent, and with suitable designs,
x-ray diffraction) to be performed in situ. In one particular
variation, the spectroscopic analysis is x-ray spectroscopy. In a
further particular variation, the x-ray spectroscopy is x-ray
diffraction.
In order to improve the performance of the device for performing in
situ x-ray spectroscopy such as x-ray diffraction and other forms
of spectroscopy where an x-ray is caused to traverse the substrate,
the number of electrons in the path of the x-ray beam of the
material being analyzed should be maximized relative to the number
of electrons that is otherwise in the path of the x-ray beam.
The number of electrons of the device that are traversed can be
reduced by choosing materials to form the device that have a low
atomic number (Z), or a low density. Examples of materials that are
preferably used for reducing the number of electrons in the
substrate material include low density plastics such as
polystyrene, polyethylene, polypropylene and other carbon based
polymers. Silicon materials, such as silicon wafers, glass,
including borosilicate and soda glass, and aerogels can be suitable
materials. Optically opaque materials that are suitable include
Beryllium, plastic films and plastics.
A key parameter R, corresponds to a ratio between the number of
electrons within the sample, (e.g., the precipitate, oil, crystal
and optionally other contents of the microvolume) that the x-ray
traverses, and the sum of the electrons contained in the support
material and the lid, or sealing material that the x-ray traverses.
##EQU1##
where the number of electrons in the x-ray beam, [e.sup.- ], is
calculated by multiplying the density of the material in grams, by
thickness of the material and the area of the x-ray beam at the
microlumen, which gives the mass in grams, X, of the microlumen
material in the x-ray beam. This can be converted into the number
of electrons by multiplying the mass in grams by Avogadro's number,
N, and dividing by the molecular weight of the material, MW. i.e.,
[e.sup.- ]=X*N/MW.
The contents of the microvolume that the x-ray beam traverses
preferably contains at least half as many electrons as is contained
in the substrate where the x-ray beam traverses. More preferably,
the portion of the microvolume that the x-ray beam traverses
contains at least one, three, five, ten times or more as many
electrons as is contained in the substrate where the x-ray beam
traverses.
In some instances, it is a particular precipitate, oil, or crystal
that is being analyzed. It is also preferred that the particular
precipitate, oil, or crystal that the x-ray beam traverses contains
at least half as many electrons as is contained in the device where
the x-ray beam traverses. More preferably, the particular
precipitate, oil, or crystal that the x-ray beam traverses should
contain at least one, three, five ten times or more as many
electrons as is contained in the device where the x-ray beam
traverses.
The number of electrons in the path of the incident x-rays can be
reduced by minimizing the mass of material in the path.
Accordingly, the substrate enclosing the microvolumes preferably
contains as little material as possible in the direction of the
path of the x-rays. As illustrated in FIG. 1A, the device housing
the microvolumes 100 will most commonly have a card shape 102 with
opposing faces 104 and 106. Walls 108, 110 (shown in FIGS. 1B and
1C) adjacent the opposing faces define a portion of the
microvolume. X-rays 112 will typically traverse the card
substantially perpendicular to the opposing faces in order to
minimize the path length across the card, that path length being
defined largely by the thickness of walls 108, 110. It is desirable
for the card to have sufficient thickness so that it will be
sufficiently rigid for necessary handling. However, by reducing the
amount of material forming the walls 108, 110 adjacent a portion of
the microvolume where x-rays will be incident, one can reduce the
amount of mass in the path of the x-rays.
FIG. 1B illustrates an embodiment where the thickness of the
overall card is reduced adjacent a region where x-rays will be
incident in order to reduce the amount of material in the path of
the x-rays.
FIGS. 1C and 1D illustrate an embodiment where less material is
present adjacent a region where x-rays will be incident in order to
reduce the amount of material in the path of the x-rays. This may
be accomplished by forming a card as shown in FIG. 1C with grooves
114 on one or both sides adjacent where x-rays will be incident. As
used herein, a groove refers to any recess formed in the substrate
so that the thickness of the device is reduced.
If the microvolume is closely adjacent one face of the card, a
groove may be formed adjacent the opposite side of the card as
shown in the cross sectional view provided by FIG. 1D. It is noted
that the card may be formed with the groove or may be formed
without the groove and then material may be removed from the card
to create the groove.
At least a portion of a groove is preferably positioned within a
lateral footprint of the microvolume where it is desired to have
the x-ray beam traverse the device. Accordingly, one embodiment of
the invention relates to a microfluidic device that comprises: a
card shaped substrate having first and second opposing faces; one
or more microvolumes at least partially defined by a first face of
the card shaped substrate; and one or more grooves at least
partially defined by a second face of the card shaped substrate;
wherein a lateral footprint of at least a portion of the one or
more grooves overlaps with a lateral footprint of at least one of
the one or more microvolumes.
At the overlap, the groove is preferably sufficiently deep that an
x-ray beam traversing the device encounters at least half as many
electrons within the microvolume as the remainder of the device
that the x-ray beam traverses. More preferably, the x-ray beam
traversing the device encounters at least one, three, five, ten
times or more as many electrons within the microvolume as the
remainder of the device that the x-ray beam traverses.
As illustrated in FIG. 1C, the microvolume may be a lumen. The
groove may have a longitudinal axis that is aligned with a
longitudinal axis of the lumen adjacent the overlapping lateral
footprint (shown in FIG. 1C). This provides a greater area for the
x-ray beam to traverse within the overlap. It is recognized,
however, that the groove may not have a longitudinal axis or may
have a longitudinal axis that is misaligned, optionally to the
extent of being perpendicular to a longitudinal axis of the lumen
adjacent the overlapping lateral footprint.
By reducing the amount of substrate encountered by an x-ray beam,
for example by using devices with grooves such as those shown in
FIGS. 1C and 1D, methods may be performed according to the present
invention comprising: performing an experiment in a microvolume of
a microfluidic device; and performing a spectroscopic analysis
using an x-ray beam that traverses the microfluidic device such
that material within the microfluidic device that the x-ray beam
traverses contains at least as many electrons as is otherwise
traversed when the x-ray beam traverses the microfluidic device.
Optionally, the material within the microfluidic device that the
x-ray beam traverses contains at least three, five, ten times or
more as many electrons as is otherwise traversed when the x-ray
beam traverses the microfluidic device.
As will be illustrated herein, crystallization conditions may be
formed by delivering different components to a single lumen or by
delivering different components to a given lumen or microchamber
from multiple different lumens. In this regard, the multiple
different lumens are preferably interconnected.
The cross sectional shape of the lumen may stay the same or may
vary along the length of the lumen. Optionally, the lumen may be
connected to one or more chambers to which material from the lumen
is delivered or from which material is delivered to the lumen. It
is noted that crystallizations may also be performed in the
chambers after material is delivered via the lumen to the
chamber.
The lumen may have a variety of cross sectional geometries. For
example, the cross-sectional geometry of the lumen may be circular,
semi-circular, ovoid, "U" shaped, square, or rectangular, or one or
more combinations thereof. Preferably, the cross sectional area of
the lumen is small relative to the length of the lumen. This serves
to reduce convection currents within liquids passing within the
lumen. Convection currents may be further reduced by the use of
thixotropic agents, such as silica gel, agarose, other
polysaccharides and polymers.
3. Layout and Use of Microsized Lumens for Performing
Crystallization Trials
Various methods and devices are provided for performing
crystallization trials in microfluidic devices. For example, in one
embodiment, a method is provided for determining crystallization
conditions for a material, the method comprising: taking a
plurality of different crystallization samples in an enclosed
microvolume, the plurality of crystallization samples comprising a
material to be crystallized and crystallization conditions that
vary among the plurality of crystallization samples; allowing
crystals of the material to form in the plurality of
crystallization samples; and identifying which of the plurality of
crystallization samples comprise a precipitate, oil or a crystal of
the material.
In another embodiment, a method is provided for determining
crystallization conditions for a material, the method comprising:
taking a plurality of different crystallization samples in a
plurality of enclosed microvolumes, each microvolume comprising one
or more crystallization samples, the crystallization samples
comprising a material to be crystallized and crystallization
conditions which vary among the plurality of crystallization
samples; allowing crystals of the material to form in plurality of
crystallization samples; and identifying which of the plurality of
crystallization samples comprise a precipitate, oil or a crystal of
the material.
In another embodiment, a method is provided for determining
crystallization conditions for a material, the method comprising:
taking a microfluidic device comprising one or more lumens having
microvolume dimensions and a plurality of different crystallization
samples within the one or more lumens, the plurality of
crystallization samples comprising a material to be crystallized
and crystallization conditions that vary among the plurality of
crystallization samples; transporting the plurality of different
crystallization samples within the lumens; and identifying a
precipitate or crystal formed in the one or more lumens.
Transporting the plurality of different crystallization samples
within the one or more lumens may be performed by a variety of
different methods. For example, transporting may be performed by a
method selected from the group consisting of electrophoresis,
electroosmotic flow and physical pumping. In one variation,
transporting is performed by electrokinetic material transport. In
a variation according to this embodiment, at least one of the
lumens optionally comprises a plurality of different
crystallization samples.
In another embodiment, a method is provided for determining
crystallization conditions for a material, the method comprising:
taking a microfluidic device comprising one or more lumens having
microvolume dimensions and a plurality of different crystallization
samples within the one or more lumens, the plurality of
crystallization samples comprising a material to be crystallized
and crystallization conditions that vary among the plurality of
crystallization samples; transporting the plurality of different
crystallization samples within the one or more lumens; and
identifying a precipitate or crystal formed in the one or more
lumens; and performing a spectroscopic analysis on the identified
precipitate or crystal while within the lumen.
The method may optionally further include forming the plurality of
different crystallization samples within the one or more lumens.
The plurality of crystallization samples may be comprised in a
single lumen or multiple lumens.
According to any of these embodiments, the methods may further
comprise forming the plurality of different crystallization samples
within the one or more lumens. The plurality of crystallization
samples may be comprised in a single lumen or a plurality of
lumens.
Also according to any of these embodiments, one or more dividers
may optionally be positioned between different crystallization
samples in the enclosed microvolumes to separate adjacent
crystallization samples.
A generalized use of a microfluidic sized lumen to form
crystallization samples and perform crystallization attempts is
illustrated in regard to FIG. 2. As shown in step A of FIG. 2, an
enclosed lumen 201 is provided such that the lumen 201 has at least
one opening 202A adjacent a first end of the lumen and at least one
opening 202B adjacent a second end of the lumen. A crystallization
experiment 203 is introduced into the lumen 201 via one of the
openings, as shown in step B. This material may be a pre-formed
crystallization experiment, consisting of a material to be
crystallized and one or more crystallization agents, or it may be a
material to be crystallized that will undergo a diffusion
experiment, wherein material will be transferred either through
vapor or liquid diffusion. Step C of the figure shows the
crystallization experiment proceeding such that a portion of the
material either crystallizes into a crystal 204 or a plurality of
crystals, microcrystals, needles, precipitates or other solids, or
the material remains in solution.
If a crystal forms, as shown in step D of the figure (shown as
205), then the crystal, precipitate, oil, etc. may be examined in
situ, for example, as shown in steps E-H. Examination may be
performed by any available method, including, but not limited to
spectroscopically, visually, or if the crystallization channel is
suitably designed, by direct exposure of x-rays. As shown by the
arrows leading from step D, the crystal or crystallization mixture
may be harvested from the lumen.
Steps E-H show different processing steps that may be performed on
the crystal or crystallization mixture. Step E illustrates a
crystal being examined within a lumen via x-ray diffraction by
using an x-ray source 208 suitable for diffraction experiments,
which is suitably focused and collimated to pass through the
material to be examined. The diffracted x-rays can then be examined
through the use of a suitable x-ray detector 206, which can be
x-ray film, one dimensional x-ray detectors, two dimensional (area)
detectors, or an electronic x-ray detector or scintillator.
Alternatively, as shown in step F, the crystal 205 can be
manipulated within the crystallization channel. This enables the
harvesting of the crystal as shown in step G, wherein the crystal
containing crystal experiment is brought to an outlet of the
crystal channel.
As shown, the crystal can be harvested into an intermediate device,
or may be harvested directly with a mounting suitable for x-ray
diffraction. This mounting can be a loop 209, as shown in step G,
or it can be a capillary suitable for x-ray mounting, or a fiber,
or a spatula. These techniques for harvesting and manipulating
crystals are widely known. Once the crystal is harvested, the
crystal can then be transported to an x-ray diffraction experiment
shown as step H where the crystal can be mounted in a position to
facilitate the diffraction experiment. It should be appreciated
that the material to be analyzed may not be a single crystal. For
example, the material may be twinned crystals, or a plurality of
crystals grouped together, or a number of loose crystals, a
precipitate, or an oil that can then be examined for crystalline
elements.
The crystallization drop 203 can be created within the microlumen,
or it can be mixed outside of the channel and introduced into the
channel. The actual method for loading the channels will vary
depending upon the necessities of the experiment. A crystallization
mixture can be formed by the use of a syringe, such as a Hamilton
syringe, or via a parallel robotic system such as the Tecam,
wherein the relevant volume of material to be crystallized is drawn
up into the syringe and then the relevant volume of the
crystallization agent can be drawn up. The material may be
dispensed directly into the loading port 202, or may be dispensed
into or onto an intermediate surface or container for mixing. The
material can be applied to the inlet port under pressure from the
syringe, or may be loaded onto the upper surface of 201, such that
the droplet covers the inlet port 202. The droplet can then be
loaded into the microlumen by the application of a pressure
difference to 202 and 202', either through pressure at 202 or
through vacuum at 202'. Similarly, after the crystal has grown, the
application of a pressure difference, either directly, or
indirectly through a pressure transfer fluid, such as mineral oil
or buffer, the crystal can be moved to the outlet port 202', for
harvesting as shown above in steps 1-3 of FIGS. 2G and 2H.
It is noted that a given lumen may have multiple lumens
interconnecting with it or extending from it. For example, as shown
in FIG. 3A, a lumen 301 may have two inlet ports 302A and 302B and
a junction 303 that may form an acute, perpendicular or obtuse
intersection. A perpendicular intersection is illustrated in FIG.
3A where the intersection 304 of channels 302C and 302D is formed
perpendicularly.
FIG. 3B illustrates how two sub-lumens extending from and joining
with a main lumen may be used to effect mixing within the main
lumen. Material 305A in one sub-lumen of the main lumen and
material 305B in the second sub-lumen extending from the main lumen
are joined and mixed together into a single volume 306 by the
geometry of the interconnecting channels. Depending upon the
particular application, the lumens and sub-lumens can be designed
to affect differing levels of mixing and the alteration of the
interface between the two substances. Obviously, the lumens may
possess both combining features or dividing features or a
combination thereof, or depending on the absolute and relative
flows combining features that function as dividing features under
altered fluid flows.
FIG. 3C illustrates the use of a dividing feature 303 to separate a
crystal containing crystallization experiment 307 into two portions
308A and 308B. It should be understood that the relative volumes in
308A and 308B may be readily attained by, suitable design or
practice, by achieving differential fluid flows.
It should also be appreciated that different combinations of single
and double ports may be combined for complex mixing, separation,
diffusion and purifications as illustrated in FIG. 3D. For example,
as shown, ports 309A and 309B meet at junction 310A. Similarly,
ports 309C, 309D meet at junction 310B. The sub-lumens from
junctions 310B and 310C can intersect at 310A.
To generate the result shown in FIG. 3A, one might apply a sample
to inlet port 302, block port 302', and apply a positive pressure
difference between port 302 and the pressure within 301. This can
be affected by applying a small vacuum at 301, by the removal of
material from 301 hydraulically, or by the application of pressure
at 302, or by the application of centrifugal force with a component
along 301. The droplet can be brought to a stop at 303 by removing
the Motive force at such a time that the material comes to rest at
304. PID (Proportional-Integrating-differential) methods and/or
controllers are very effective for optimizing fluid delivery
accounting for hysteresis effects within the fluid transfer
mechanisms and the microlumens.
In FIG. 3B, material can be applied at 302 and 302' and then
individually advanced as described above, or may be advanced in
tandem by the application of pressure differential across both
fluids simultaneously to yield the combined mixture 306 at the
union of the two microlumens.
FIG. 3C is constructed by inducing the material assembled in a
crystallization bolus 306 to crystallize. Pressure can then be
applied to the interior of the microlumen 301 to force the crystal
containing bolus 307 along the microlumen 301 to the intersection
303. This pressure can be applied hydraulically to port 302 or
302', while sealing the other, or to both ports 302 and 302'
simultaneously. The hydraulic pressure can be applied directly via
syringe or syringe pump or via a hydraulic transfer fluid such as
water or mineral oil using a fluid filled syringe or syringe pump,
with or without a connecting manifold to facilitate the application
of the hydraulic pressure to the ports 302 and 302'. At harvest, by
modulating the pressure difference between two outlet ports, the
unwanted crystallization liquor can be preferentially forced into
the waste passage as bolus 308', while concentrating the crystal in
a desired amount of crystallization liquor in bolus 308'. The
pressure can be modulated by differential pumping of two syringe
pumps connected to the respective outlet ports. This can be done
under manual control with a simple joystick controller, or it can
be accomplished with computer vision software, such as that
provided by Keyance.
The methods and the devices of the present invention will now be
described with regard to the following figures.
FIGS. 4A-4C provide several embodiments of performing
crystallizations within a lumen. It should be recognized that
depending upon the differing surface energies of the solutions and
the enclosure the actual interfaces may be convex, concave, flat or
with elements of all three. For illustrative purposes, the
interfaces are shown to be spherical.
FIG. 4A illustrates a crystallization mixture 401 formed within a
lumen 403 positioned between two dividers 400, 402. The lumen 403
is formed at least partially by a substrate 407 and enclosed
therein.
Generally, dividers may be used according to the present invention
to separate aliquots of material within the microvolumes, typically
the lumens. The separated aliquots of materials may correspond to
separate experiments such as the crystallization trials described
herein.
The dividers 400, 402 may be semi-permeable gases or liquids,
semi-permeable gels or permeable gels, or thixotropic liquids, or
immiscible and impermeable liquids or beads. As a result, the
interface formed between a crystallization and a divider may be a
liquid/liquid, liquid/gas interface, liquid/solid or liquid/sol-gel
interface. In some instances, the interface may also be a membrane,
gel, frit, or matrix to modulate or alter the diffusion
characteristics.
The dividers can be impermeable, semi-permeable or permeable. For
example, semi-permeable substances such as air, oil, solvent, gel
and beads can be used as dividers. The dividers can also be
physical constructions, such as a narrow pore, a thin passage, a
frit or sintered beads or powders.
In one variation, the dividers function to modulate diffusion
characteristics between adjacent samples. For example, the one or
more dividers may be formed of a semi-permeable material that
allows diffusion between adjacent crystallization samples.
In crystallizations performed within lumens according to the
present invention, there may be one or multiple crystallization
conditions, either related or unrelated in a given lumen. The
dividers serve to separate and optionally isolate the different
crystallization conditions. For example, a second crystallization
condition, potentially one of many, is illustrated by dividers 404,
406 surrounding crystallization 405. The dividers and the gap 403
may optionally be omitted.
Alternatively, the substance to be crystallized can be element 401,
with 400 and 402 being crystallization agents, either identical or
different. In this instance, element 403 functions as a barrier
between one condition and the next. As illustrated in FIG. 4B,
element 403 can be omitted for a series of crystallizations.
By positioning barrier material on opposing sides of a
crystallization within the microlumen, the crystallization may be
encased and its length thereby controlled. Examples of barrier
materials that may be used include, but are not limited to
immiscible solvents or solids. The barrier materials may form a
complete or partial barrier. Complete barriers prevent the
crystallization from traversing the barrier material. Partial
barriers limit the rate at which components of the crystallization
traversing the barrier material. Examples of partial barrier
materials include, but are not limited to polymers or solvents that
allow for diffusion. Diffusion within the crystal conditions can be
further modified by the use of thixotropic agents, gels or sols to
prevent convective movements of the solutions.
As an alternative method, the substance to be crystallized may be
elements 400, 402, 404, and 406. In this instance, elements 401 and
405 may be crystallizing agents. Element 403 meanwhile may be a
barrier or can be another crystallization agent.
In all cases, the crystallization agent can be mixed prior to
attempting to perform the crystallization within the lumen or can
act in situ, with no prior mixing.
FIG. 4B illustrates a crystallization performed within a lumen
where multiple crystallization conditions are simultaneously
employed. As illustrated, elements 411, 413, and 415 can be
crystallization conditions, either premixed with crystallization
agents or not. If elements 411, 413, and 415 are premixed, then
elements 410, 412, 414, 416 may optionally be a semi-permeable gas
or liquid, a semi-permeable gel or permeable gel, a thixotropic
liquid, an immiscible liquid, an impermeable liquid or bead, or a
crystallization agent.
In the instance where 411, 413, and 415 are not premixed, then
minimally, 412 and 414 are crystallization agents and the termini,
410 and 416 are a barrier (e.g., either a bead, an impermeable
substance or a gas bubble).
In the instance that the crystallization is rapid, it is not
necessary to have impermeable termini. Instead, diffusion from the
termini can be used as an additional crystallization agent.
FIG. 4C illustrates a crystallization performed within a lumen
where a series of crystallization agents are set up for
crystallization against a series of substances to be crystallized.
In FIG. 4C, the crystallization agents are shown as elements 420,
422, 424, and 426. The crystallization attempts comprising
substances to be crystallized are shown as elements 421, 423, and
425. These crystallization attempts may or may not be
identical.
The sequential crystallizations can be formed in the microlumen by
the sequential addition of the materials in inverse order. Thus,
sample 406 may be loaded into the microlumen, followed by 405,
followed by 404 and so forth. Obviously, the microlumen can be
loaded from right to left or left to right. The individual
crystallizations may be made on the cassette by using a manifold
such as the one show in FIG. 3D, and then varying the relative
pressures in the manifold individually or in parallel to achieve
the desired mixing. For instance, barrier material might be loaded
via 309, protein via 309', semi-permeable material via 309", and a
crystallization agent via 309'". The alternating volumes of fluid
can be easily made outside of the microlumen by the sequential
loading of a syringe pump. To do this, the syringe loads the first
sample volume from the source of the first material 400 by creating
a pressure differential. The second material 401 is then loaded by
the same method. Then the next material 403 is loaded until either
the volume limit is reached upon the syringe or the desired
contents of the microlumen have been loaded. The syringe pump can
then unload the contents into the inlet port on the microlumen.
FIG. 4C might be conveniently constructed through the use of a Tee
as shown in FIG. 3A, wherein a series of crystallization conditions
could be injected into the microlumen, alternating with suitable
injections of material to be crystallized. It will be appreciated
by those skilled in the art, that complete droplets can be made by
small bursts of differential pressure.
FIGS. 5A-5D illustrate crystallizations being performed within
lumens where one or more of the elements of the crystallization
experiment change along a length of the lumen. As will be
explained, the change can occur discretely or continuously, and
need not be changed in a simple linear method.
FIG. 5A illustrates a lumen 501 where the crystallization condition
is different across the lumen.
FIG. 5B illustrates a series of substances to be crystallized,
shown as elements 510, 511, 512, and 514. These substances are
present in a single gradient 513 such that the different elements
are exposed to different crystallization conditions.
FIG. 5C illustrates an alternative to the embodiment shown in FIG.
5B. In this embodiment, a series of different crystallization
agents 520, 521, 522, and 524 are present within the lumen and are
used to provide different conditions for crystallizing substance
523 present across the lumen.
FIG. 5D illustrates diffusion between the various elements in an in
situ crystallization. Termini 1 and 7 share a single interface for
diffusion. Each of the remaining portions of the in situ
crystallization share at least two distinct interfaces for
diffusion. Thus, a single substance to be crystallized, present
across the lumen, can be assayed against two or more
crystallization agents simultaneously. For example, substance 2 is
shown to share two separate interfaces, which can cause crystals to
grow either near the 1 to 2 interface or the 2 to 3 interface.
Crystals growing in the center of 2 are indicative of a substance
that requires aspects of both 1 and 3 to crystallize.
The gradient shown in FIG. 5A can be created by using a "Tee" shown
in FIG. 3A together with a series of mixing baffles downstream.
Initially all of the input flow comes from one of the ports, for
example 302". The flow in the second port 302'" is increased,
usually with a corresponding decrease in the amount of material
flowing in at 302". The relative injection volumes, the total
volume injected and the rate at which they change will affect the
final gradient produced. Gradients may be formed off chip by
similar means. The addition of a series of crystallization agents
can be effected via the use of a "Tee" as described above, or may
be individually loaded in an inlet port.
FIG. 6A illustrates a crystallization performed within a lumen
where a single crystallization condition 601 occupies an entire
crystallization space.
FIG. 6B illustrates multiple crystallizations being performed
within a lumen where dividers 611, 612, 615, 617 are used between
the crystallizations 610, 612, 614, 616, and 618. The dividers are
shown in the figure to have planar surfaces adjacent the
crystallizations.
FIG. 6C illustrates multiple crystallizations being performed
within a lumen where dividers 621, 623, 625 are used between the
crystallizations 622, 624. The dividers are shown in the figure to
have curved, convex surfaces adjacent the crystallizations 622, and
624 that have complementary concave surfaces. The actual shape of
the meniscus dividing the samples is a function of the surface
tension at the interface and the surface of the microlumen.
FIG. 7A shows a device for performing a series of crystallizations
within a series of lumens where each lumen comprises a loading port
701 and an unloading port 703 and a lumen body 702 interconnecting
the ports.
FIG. 7B shows a cross section of a device 700 for performing a
crystallization within a lumen where the lumen 702 is not
enclosed.
FIG. 7C shows a cross section of a device 700 for performing a
crystallization within a lumen where the lumens 702 are rectangular
in shape.
FIG. 7D shows a cross section of a device 700 for performing a
crystallization within a lumen where the lumens 702 are curved or
tubular in shape.
FIG. 7E shows a device for performing crystallizations within a
series of lumens where the lumens are loaded with samples 705, 707
that are separated by divider or modifier segments 704, 706, 708.
It should be appreciated that each discrete sample may have
conditions that are potentially unique and unrelated to adjacent
samples. The dividers or modifiers positioned between the samples
can be permeable, semi-permeable or impermeable.
The series of crystallizations shown in 7E can be created via the
same methods used to create samples 4A above. To expedite the
process, it is preferable to load some or all of the channels
simultaneously.
FIG. 8A shows a device 800 for performing a series of different
crystallizations within a series of lumens where each lumen
comprises a loading port 801 and unloading port 803 and a lumen
body 802 interconnecting the ports.
FIG. 8B illustrates a single lumen 802 in which a barrier 805 is
adjacent to the crystallization condition 806 bounded by second
barrier 807. The crystallization conditions can be a larger volume,
or the same volume or smaller volume than the barriers. A more
complex form of FIG. 8B, is shown in FIG. 8C. It is noted that the
barriers used herein correspond to the dividers described
above.
FIG. 8C illustrates a diffusion crystallization. The barrier 805',
which can be either permeable or impermeable, is adjacent to the
crystallization condition 806', which is bounded by the barrier
807', which can be either permeable, semi-permeable and is adjacent
to crystallization condition 808', which differs from condition
806' in at least one component. Condition 808' is bounded by
boundary condition 809', which can be permeable, semi-permeable or
impermeable. Conditions 806' and 808 form a set of linked
crystallization conditions, whose rate of equilibration is
modulated by the properties of barrier 807'. This example can be
easily generalized to an entire crystallization channel or plate by
suitable construction of the conditions and the plate itself. This
is illustrated with regard to FIG. 8D.
FIG. 8D illustrates a multi-component crystallization being
performed in a single lumen. The multi-component crystallization
consists of end barriers 809 and 839 and crystallization conditions
811 through 837, each separated from its adjacent neighbor
conditions by a permeable or semi-permeable barrier 810, repeating
along the channel as 810' between each condition 811 through 837.
Any number of conditions can be coupled via semi-permeable or
permeable barriers depending on the dimensions of the lumen, the
design of the plate and crystal arrays and the volumes of the
various crystal conditions.
Various methods may be performed using devices that allow for
diffusion between adjoining flows within a single lumen. For
example, in one embodiment, a microfluidic method is provided that
comprises: delivering first and second fluids to a lumen of a
microfluidic device such that the first and second fluids flow
adjacent to each other within the lumen without mixing except for
diffusion at an interface between the first and second fluids,
wherein the first fluid is different than the second fluid. In a
variation according to this embodiment, the composition of at least
one of the first and second fluids is varied over time as it is
delivered to the lumen so that the fluid forms a gradient with
regard to a concentration of at least one component of the fluid
that changes along a length of the lumen. In another variation
according to this embodiment, the microfluidic device may comprise
a plurality of lumens, the method further comprising delivering
first and second fluids to each of the plurality of lumens.
According to this embodiment, the same first and second fluids may
be delivered to each of the plurality of lumens. Alternatively,
different first and second fluids are delivered to the different
lumens of the plurality of lumens. The first and second fluids may
also have a same or different flow rate within the lumen.
In another embodiment, a microfluidic method is provided that
comprises: delivering first and second fluids to a lumen of a
microfluidic device such that the first and second fluids flow
adjacent to each other within the lumen without mixing except for
diffusion at an interface between the first and second fluids,
wherein the first fluid is different than the second fluid and a
composition of at least one of the first and second fluids
delivered to the lumen is varied so that the composition of at
least one of the first and second fluids within the lumen varies
along a length of the lumen.
In yet another embodiment, a microfluidic method is provided that
comprises: delivering first, second and third fluids to a lumen of
a microfluidic device such that the first, second and third fluids
flow adjacent to each other within the lumen without mixing except
for diffusion at an interface between the first, second and third
fluids, wherein the first, second and third fluids are different
than each other and a composition of at least one of the first,
second and third fluids delivered to the lumen is varied so that
the composition of at least one of the first, second, and third
fluids within the lumen varies along a length of the lumen.
According to any of these embodiments, the composition of at least
one of the first, second and third fluids may be varied over time
as it is delivered to the lumen so that the fluid forms a gradient
with regard to a concentration of at least one component of the
fluid that changes along a length of the lumen. Also according to
any of these embodiments, the microfluidic device may comprise a
plurality of lumens, the method comprising delivering first, second
and third fluids to each of the plurality of lumens. The same or
different first, second and third fluids may be delivered to each
of the plurality of lumens. Optionally, at least one of the first,
second and third fluids have a different flow rate than another of
the fluids within the lumen. Also, at least one of the first,
second and third fluids may have the same flow rate than another of
the fluids within the lumen. Also according to any of these
embodiments, the first, second and third fluids may be combined to
form different crystallization conditions for crystallizing a
molecule such as a protein. In one variation, the first, second and
third fluids combine to form different crystallization conditions,
the second fluid comprising the material to be crystallized and
being positioned between the first and third fluids.
In regard to any of these embodiments, dividers may optionally be
used in one or more of the first, second and optionally third or
more fluids. These dividers may be used to set up multiple separate
aliquots in the fluid flow where the dividers are positioned.
One particular application of these various methods is the use the
first, second and optionally third or more fluids to form different
crystallization conditions for crystallizing a material such as a
protein.
Devices and methods that allow for diffusion between adjoining
flows within a single lumen will now be described in regard to
FIGS. 9A-9D.
FIG. 9A shows an embodiment of a device 900 being used to perform a
series of different crystallizations within a series of multi-lumen
assemblies 901, 901' where each multi-lumen assembly comprises at
least one and preferably two loading 902, 903 and unloading 907,
908 ports and a lumen body 901 interconnecting the ports.
The fluids for each crystallization may be contained in two
distinct fluid flows 902, 903 from the port that are in contact
with each other along a shared interface 906.
It is noted that this shared interface 906 is not a structure but
is an interface that forms between the two distinct fluid flows as
a result of laminar flow within a microvolume dimensioned lumen. By
contrast, FIGS. 10A-10B describe adjacent fluid flows in separate
lumens where a permeable or semi-permeable shared wall is
positioned between the lumens that allows for diffusion between
fluids in the separate lumens.
The fluid flows consist of a crystallization condition 909 and a
series of crystallization conditions 904 and 904' that are
separated by a barrier 905, which may be permeable, semi-permeable
or impermeable. This arrangement enables the simultaneous
examination of many different conditions against a single
condition.
The lumen shown in FIG. 9A can be either preloaded with a fluid or
not. It is preferable, however, that the pairs of fluid flows be
simultaneously loaded via the inlet ports 902 and 903. Having an
existing fluid in the lumen may facilitate maintaining the laminar
flow necessary to maintain a uniform interface 906 between the two
fluid flows.
A method for loading a single channel has been described above.
This process can be used to produce the samples introduced via
inlet port 902. Simultaneous with the injection of the material via
port 902 is the injection of the desired material 909. This method
can be easily generalized to more than two fluid flows.
FIGS. 9B-9D show an embodiment of a crystallization experiment that
may be performed using the device of FIG. 9A.
FIG. 9B illustrates an enlargement of a lumen of the device shown
in FIG. 9A which illustrates some of the different simultaneous
diffusions that are made possible by the invention. In fluid flow
910, aliquots of crystallization agents 912, 914 are positioned on
opposing sides of permeable or semi-permeable barrier 916. Further
aliquots may be positioned upstream and downstream of the portion
of the flow shown. Barriers 918, 920 on the outer sides of aliquots
912, 914 may be impermeable and thereby isolate these aliquots
relative to the remainder of the fluid flow. As shown, barriers
918, 920 are permeable or semipermeable, allowing for diffusion
further along fluid flow 910.
As illustrated in FIG. 9C, when the intervening barriers are
permeable or semi-permeable, diffusion occurs through the barrier
to form a gradient from condition 912 to condition 914.
Furthermore, fluid flow 922 adjacent fluid flow 910 also forms a
diffusion front between the two flows. As a result, diffusion
between fluid flow 922 and aliquots 912, 914 also occurs. These
different diffusions are illustrated in FIG. 9C by circles 924,
926, 928 and 930.
As can be seen from FIG. 9C, this method provides for the diffusion
of crystallization components longitudinally between differing
conditions in a manner that can be regulated through the suitable
choice of barriers as well as laterally across an interface formed
by the laminar flow of microfluid flows.
FIG. 9D illustrates crystals forming at different locations. For
example, crystal 932 is shown positioned between the fluid flows
and crystal 934 is shown positioned within divider 916. Meanwhile,
crystal 936 is shown to be formed on the portion of the diffusion
interface farthest from aliquot 914. The positioning of the
crystals relative to the aliquots and the fluid flows can be used
to indicate which crystallization conditions are conducive and not
conducive to crystal formation. As can be seen, the multiplicity of
different conditions that may be formed by using these multiple
different diffusion fronts allows a great level of diversity of
crystallization conditions to be created.
Crystallization conditions can be examined as part of a time series
and the time and location of nucleation, initial crystallization or
precipitation can be observed or derived. By using known, or
observed diffusion rates, the actual conditions at the nucleation
or crystallization points can be determined and used for further,
more detailed crystallizations. This method thus allows for a finer
and more complete examination of crystallization conditions than
can be afforded by single condition mixing of crystallization
agents and the material to be crystallized.
The diffusions illustrated in FIGS. 9B-9D are based on diffusion
between first and second adjacent fluid flows. It should be
recognized that this method and device design can be readily
extended to three, four, or more adjacent fluid flows.
The diffusions illustrated in FIGS. 9B-9D are also based on
multiple aliquots in the first fluid flow. These multiple aliquots
may have the same or different composition. They may comprise
crystallization agents and/or the material to be crystallized It
should be recognized that the second fluid flow may also have
multiple aliquots.
Various devices and methods are also provided that allow for
diffusion between adjoining lumens. For example, in one embodiment,
a microfluidic device is provided that comprises: a substrate; a
first lumen at least partially defined by the substrate; and a
second lumen; wherein the first and second lumens share a common
wall with each other that allows for diffusion between the two
lumens over at least a portion of the length of the two lumens.
In another embodiment, a microfluidic device is provided that
comprises: a substrate; a plurality of sets of lumens, each set
comprising a first lumen at least partially defined by the
substrate, and a second lumen, wherein the first and second lumens
share a common wall with each other that allows for diffusion
between the two lumens over at least a portion of the length of the
two lumens.
It is desirable for these devices to allow for a high degree of
parallel experimentations. Accordingly, the devices preferably
comprise at least 4, 8, 12, 24, 96, 200, 1000 or more sets of
lumens with adjoining walls.
According to each embodiment, the common wall may optionally
comprise a membrane, gel, frit, or matrix that allows for diffusion
between the two lumens.
Also according to each embodiment, the device may further comprise
a third lumen, the third lumen sharing a common wall with at least
one of the first and second lumens so as to allow for diffusion
between the lumens over at least a portion of the length of the
lumens.
Microfluidic methods are also provided. For example, in one
embodiment, a microfluidic method is provided comprising:
delivering a first fluid to a first lumen of a microfluidic device
and a second, different fluid to a second lumen of the microfluidic
device, the first and second lumens sharing a common wall that
allows for diffusion between the lumens over at least a portion of
the length of the lumens; and having the first and second fluids
diffuse between the first and second lumens.
It is noted in regard to the devices and methods that laminar flow
allows for separate fluid flows to be delivered in a same lumen, as
discussed above in regard to FIGS. 9A-9D. As a result, it should
also be recognized that the common wall need not be present along
an entire length of the adjacent lumens. Further, it should be
noted that one, two or more separate fluid flows may be added to
each lumen.
Devices and methods that allow for diffusion between adjoining
lumens will now be described in regard to FIGS. 10A-10B
FIG. 10A illustrates an embodiment of a device 1000 being used to
perform a series of different crystallizations within a series of
multi-lumen assemblies 1001, 1001' where each multi-lumen assembly
comprises a first lumen 1002 and a second lumen 1004, the first and
second lumens sharing a common wall 1006 that allows for diffusion
between the lumens over at least a portion of the length of the
lumens. Each lumen has a separate loading 1008, 1010 and unloading
1012, 1014 ports that are each in fluid communication with a
lumen.
A separate fluid flow 1016, 1018 is delivered to each lumen through
the loading ports 1008, 1010.
It is noted that the common wall 1006 in FIG. 10A differs from the
shared interface 906 of FIG. 9A because it is an actual permeable
or semi-permeable structure positioned between the lumens that
allows for diffusion between fluid in the separate lumens.
FIG. 10B illustrates an alternate embodiment where the multi-lumen
assembly comprises a first lumen 1002, a second lumen 1004, and a
third lumen 1005 where the first, second and third lumens share
common wall 1006, 1007 that allows for diffusion between the lumens
over at least a portion of the length of the lumens.
It is noted that a composition of at least one of the first and
second fluids may be varied so that the composition of at least one
of the first and second fluids varies along a length of the lumen.
The composition of at least one of the first and second fluids may
also vary over time as it is delivered to the lumen so that the
fluid forms a gradient with regard to a concentration of at least
one component of the fluid that changes along a length of the
lumen. Depending on the experiment, the same or different first and
second fluids may be delivered to each of the plurality of first
and second lumens.
It is also noted that the first and second fluids may have a same
or different flow rate within the lumen.
When a third separate lumen is provided, the method may optionally
further comprise delivering a third fluid to a third lumen which
shares a common wall with at least one of the first and second
lumens, the common wall allowing for diffusion between the third
lumen and the first or second lumen over at least a portion of the
length of the lumens.
It should be recognized that the embodiments of FIGS. 9A-9D and
FIGS. 10A-10B may optionally be combined. For example, a device
according to FIG. 10A or 10B may be employed where two or more
fluids flows are introduced into a single lumen, as illustrated in
FIGS. 9A-9D.
FIGS. 10C-10E show an embodiment of a crystallization experiment
that may be performed. Shown in the figures are an enlargement of
the double lumen device shown in FIG. 10A. Lumen 1002 is separated
from lumen 1004 by the permeable or semi-permeable wall 1006.
In FIG. 10C, lumen 1002 is illustrated as containing a series of
crystallization agents 1020, 1020', and 1020". These
crystallization agents in lumen 1002 are separated by impermeable
dividers 1022' and a semi-permeable divider 1022. This allows
separate aliquot to be created in that lumen. It should be
recognized that the dividers employed in this embodiment are
optional.
Lumen 1004 meanwhile is shown to contain the mixture to be assayed
for crystallization, such as a protein solution in a buffer. Once
the lumens are filled, diffusion between the differing chemical
mixtures begins.
As shown in FIG. 10D, after some time has passed, diffusion can
occur both through the semi-permeable internal divider 1022, as
well as through the permeable or semi-permeable wall 1006. The
chemical gradients from the crystallization agents are illustrated
as diffusion fronts 1024, 1026, diffusing into the crystallization
mixtures, and as the intra-lumen diffusion from 1021. It is noted
that diffusion front 1021 can also permeate through the permeable
or semi-permeable wall 1006, resulting in a joint diffusion front
1025.
FIG. 10E illustrates a sample series of crystal growths that have
occurred after some diffusion has occurred. Crystal 1028 is in the
center of diffusion front 1024 and has resulted largely from the
action of crystallization condition 1020'. Crystal 1029 has grown
on the diffusion interface 1025 and is therefore indicative of a
crystal that needs chemical moieties of both condition 1020' and
condition 1020" to form. In contrast, crystal 1030 has formed on
the portion of the diffusion interface farthest from condition
1020', suggesting that some aspect of condition 1020' slows or
prevents crystal growth in the context of the crystallization agent
1020". As can be seen, the multiplicity of different conditions
that may be formed by using these multiple different diffusion
fronts allows a great level of diversity of crystallization
conditions to be created.
As shown in FIG. 10F, multiple lumens 1002, and 1004 may be
separated by permeable or semi-permeable wall 1006. Lumens 1004 and
1005 are separated by permeable or semi-permeable wall 1007. By the
suitable loading of crystallization conditions, 1040, 1041, 1042,
1043, 1044, 1045, 1046, 1047, 1048, and 1049, the skilled artisan
can produce many well defined opportunities for diffusion between
the various conditions. By the design of suitable experiments the
diffusion rates of the chemical moieties within crystallization
conditions can be determined. Once the diffusion patterns have been
established, the location of crystals within the lumen can be used
to interpolate the nucleation and crystal growth conditions between
the existing conditions.
FIG. 11A illustrates a single lumen with integral mixing and
harvesting channels. The single channel comprises an inlet assembly
of at least two inlet ports 1102, 1103 and mixing channel 1104, a
crystallization channel 1101 and a harvesting assembly 1107,
comprised of at least one harvesting port 1105 and preferably two
ports 1106 for harvesting.
FIG. 11B shows an embodiment of a device 1100 for performing a
series of different crystallizations within a series of lumens
1101, 1108-1114 where each lumen comprises integral mixing and
harvesting channels. Conditions with each channel may consist of
identical conditions, or of multiple crystallization agents or of
multiple substances to be crystallized or any combination
thereof.
The "Y" shown in FIGS. 11A and 11B is easily utilized to alternate
a series of materials. Syringes or syringe pumps can alternately
deliver material to ports 1102 and 1103. Simply, a small
interruptible vacuum can be applied to either 1105 or 1106 and the
other can be sealed. Alternatively, the vacuum can be applied to
both. Whenever a sample is loaded into 1102, port 1103 is sealed
and the vacuum is applied at 1105/1106 to transfer the appropriate
volume into the "Y" 1104. When the desired volume has been
transferred, the pressure differential is removed. Material can
then be loaded at 1103, port 1102 is then sealed and a pressure
difference sufficient to deliver the required volume into 1104 is
applied. The process can then be repeated. For ease of control, it
may be preferable to preload the lumens with a hydraulic transfer
fluid. Similarly, it is preferable to apply constant pressure
difference between the pair 1102/1103 and the pair 1105/1106. The
lumen 1101 can then be loaded by alternating the supply of material
from 1102 and the supply of material from 1103.
FIG. 12A illustrates a device 1200 comprising a series of lumens,
each lumen having attached to it an array of individual
crystallization cells 1204, each cell having at least one separate
inlet 1202 or outlet 1203 and at least one channel connecting the
cell to the lumen 1201. Each crystallization cell may have an
exclusive inlet and outlet, giving an array of independent cells,
or the cells may be linked, or multiplexed with a common inlet or
outlet lumen 1201, which can have a single 1202 or multiple ports
1202, 1205. Substances unique to each crystallization cell are
loaded via the port 1203, with the excess being drawn off via the
common lumen. Substances common to all cells in a sub-array 1208,
consisting of port 1202, manifold 1201, port 1205 and all thus
linked crystallization cells and ports, can be loaded through ports
1202 and 1205 by any combination of injection of suction via the
ports within the sub-array. By suitable application of driving
forces, substances can be driven into any one of the attached
crystallization cells, either in parallel or individually.
FIG. 12B illustrates an embodiment of an individual crystallization
cell shown in FIG. 12A. If the device is to consist of individually
accessed crystallization cells, then port 1201 is unique to each
cell. If the cassette includes multiple sub-arrays, then lumen 1201
may be common with the other crystallization cells of the
sub-array.
FIG. 12C illustrates an embodiment of an individual crystallization
cell 1204 shown in FIG. 12A where the cell comprises a
crystallization agent 1207 and a substance 1206 to be crystallized.
The exact nature of the meniscus between the substances is highly
dependent upon both the sequence of addition of the crystallization
materials, their relative volume and the surface properties of the
supporting materials of the cassette, e.g. surface energy,
hydrophobicity, hydrophilicity, or adsorbed materials.
4. Delivery of Materials to Microsized Lumens
Materials may be added to the devices of the present invention by a
variety of different methods and mechanisms. For example, material
may be added to a given lumen by the sequential addition of the
volumes of materials that need to be added or may be delivered as a
single bolus. Commercial robots such as the Staubli can deliver
small volumes of material with the high degree of accuracy needed
to repeatedly deliver the necessary drops into entry ports of the
lumens. For improved accuracy, multiple deliveries can be used to
create the final, larger volume, from a series of smaller
volumes.
Volume of materials can be delivered by a number of different
mechanisms, such as ultrasonic dispensers, peristaltic pumps,
syringes, syringe pumps or stepper motor driven plungers. Materials
can be delivered to multiple different lumens individually, in
parallel within a channel, or in parallel across the entire device.
For some embodiments, it may be desirable to deliver two or three
conditions simultaneously for optimal loading. Alternatively, it is
possible to use pin arrays to deliver the fluid.
The device may also be docked with a manifold in order to deliver
materials to the lumens of the device. This manifold can be mated
with at least one or multiple inlet ports. The channels in the chip
can then be filled individually, or in parallel from the manifold.
The filling cycle may be entirely in parallel, or the filling cycle
may involve multiple docking events. If the device is docked
multiple times to different manifolds, the materials can be added
by alternately mating the device with, for example, a protein
manifold, a barrier manifold, and a crystallization manifold. The
materials may be pressure driven into the device, or may be applied
with vacuum, or a combination thereof. Under some constructs, it
may be advantageous to pre-fill the device with a fluid. This fluid
can then be displaced by the pressure addition of material, or this
fluid may be removed actively be an applied vacuum, or a
combination thereof to deliver the necessary fluids. Pre-filling
the device has advantages in the fluidics, and also for the
alteration or modulation of the surface properties of the
lumen.
5. Transport in Microfluidic Devices Using Centrifugal Force
One feature of the present invention relates to the use of
centrifugal force to cause material to flow within the microlumens
of devices according to the present invention. Through the use of
centrifugal force, fluids can be loaded, measured, filtered, mixed
and incubated within a lumen. The centrifugal force serves to
generate hydrostatic pressure to drive the fluids through the
lumens, reservoirs, filters and manifolds. This method has the
advantages of speed, tightly enclosed fluids to minimize
evaporation, and simplicity since there are no moving parts on the
device to break or become fouled through the application of
external energies. The use of centrifugal force is compatible with
a wide variety of fluids.
Optionally, the centrifugal forces are applied such that at least
0.01 g, 0.1, 1 g, 10 g, 100 g or more force is applied to the
material in the device to cause the material to move within the
microvolumes.
Applying the centrifugal forces may be performed by rotating the
device. Optionally, the centrifugal forces are applied by rotating
the device at least 10 rpm, 50 rpm, 100 rpm or more. It is noted
that the rotational axis about which the microfluidic device is
rotated may be positioned within or outside the lateral footprint
of the microfluidic device.
A particular advantage of the use of centrifugal force is the
ability to make hundreds to thousands to hundreds of thousands of
replicate volume measurements simultaneously. Accordingly, devices
may be designed so that material in at least 4, 8, 12, 36, 96, 200,
1000 or more different microvolumes are transported when
centrifugal force is applied.
In addition, since a common amount of force can be applied to each
lumen and the shapes of the lumens can be closely controlled,
replicate volume measurements can be made with a high degree of
reproducibility. For example, devices can be designed so that the
volume of fluid delivered from in a given microvolume upon the
application of centrifugal force is within 50%, 25%, 10%, 5%, 2%,
1% or less of the volume of fluid delivered in any other
microvolumes.
A further feature of the use of a device employing centrifugal
force is the ability to preload crystallization agents. This can be
used to dramatically enhance the speed and efficiency of the
crystallization setup.
In one embodiment, a microfluidic method is provided that
comprises: taking a microfluidic device comprising a plurality of
microvolumes; and causing movement of material in a same manner
within the plurality of microvolumes by applying centrifugal forces
to the material.
In another embodiment, a microfluidic method is provided that
comprises: taking a plurality of microfluidic devices, each device
comprising a plurality of microvolumes; and causing movement of
material in a same manner within the plurality of microvolumes of
the plurality of devices by applying centrifugal forces to the
material.
In a variation, the plurality of microfluidic devices may be
stacked relative to each other when the centrifugal forces are
applied. The plurality of microfluidic devices may also be
positioned about a rotational axis about which the plurality of
microfluidic devices are rotated to apply the centrifugal
forces.
In another embodiment, a microfluidic method is provided that
comprises: taking a microfluidic device comprising a plurality of
microvolumes; and physically moving the device so as to effect a
same movement of material within the plurality of microvolumes.
Physically moving the device preferably causes centrifugal force to
be applied, for example, by rotation of the device about an axis.
According to this embodiment, the material moved in each of the
plurality of microvolumes by movement of the device preferably has
a same quantity.
In another embodiment, a microfluidic method is provided that
comprises: taking a microfluidic device comprising a plurality of
microvolumes; and accelerating or decelerating a motion of the
device so as to effect a same movement of material within the
plurality of microvolumes. According to this embodiment, the motion
of the device is optionally a rotation of the device. In such
instances, acceleration or deceleration may be caused by a change
in a rate of rotation of the device.
In another embodiment, a microfluidic device is provided that
comprises: a substrate; and a plurality of microvolumes at least
partially defined by the substrate, each microvolume comprising a
first submicrovolume and a second submicrovolume that is in fluid
communication with the first submicrovolume when the device is
rotated, the plurality of microvolumes being arranged in the device
such that fluid in the first submicrovolumes of multiple of the
microvolumes are transported to second submicrovolumes of the
associated microvolumes when the device is rotated.
In yet another embodiment, a microfluidic device is provided that
comprises: a substrate shaped so as to provide the device with an
axis of rotation about which the device may be rotated; and a
plurality of microvolumes at least partially defined by the
substrate, each microvolume comprising a first submicrovolume and a
second submicrovolume that is in fluid communication with the first
submicrovolume when the device is rotated, the plurality of
microvolumes being arranged in the device such that fluid in the
first submicrovolumes of multiple of the microvolumes are
transported to the second submicrovolumes of the associated
microvolumes when the device is rotated about the rotational axis.
Optionally, the second microvolumes are lumens.
The device may optionally comprise a mechanism that facilitates the
device being rotated about the rotational axis. For example, the
substrate may define a groove or hole at the rotational axis that
facilitates the device being rotated about the rotational axis.
Optionally, a center of mass of the device is at the rotational
axis and the substrate defines a groove or hole at the rotational
axis that facilitates the device being rotated about the rotational
axis. In one variation, the device is disc shaped, the substrate
defining a groove or hole at the rotational axis of the disc that
facilitates the device being rotated about the rotational axis.
In another embodiment, a microfluidic method is provided that
comprises: taking a microfluidic device comprising a substrate, and
a plurality of microvolumes at least partially defined by the
substrate, each microvolume comprising a first submicrovolume and a
second submicrovolume where the first submicrovolume and second
microvolume are in fluid communication with each other when the
device is rotated; adding fluid to a plurality of the first
submicrovolumes; and rotating the device to cause fluid from the
plurality of first submicrovolumes to be transferred to the second
submicrovolumes in fluid communication with the first
submicrovolumes.
In another embodiment, a microfluidic method is provided that
comprises: taking a plurality of microfluidic devices, each
comprising a substrate, and a plurality of microvolumes at least
partially defined by the substrate, each sample microvolume
comprising a first submicrovolume and a second submicrovolume where
the first submicrovolume and second submicrovolume are in fluid
communication with each other when the device is rotated; adding
fluid to a plurality of the first submicrovolumes in the plurality
of microfluidic devices; and rotating the plurality of microfluidic
devices at the same time to cause fluid from the plurality of first
submicrovolumes to be transferred to the second submicrovolumes in
fluid communication with the first submicrovolumes.
In another embodiment, a microfluidic method is provided that
comprises: taking a microfluidic device comprising a substrate, and
a plurality of microvolumes at least partially defined by the
substrate, each microvolume comprising a first and a second
submicrovolume where the first and second submicrovolumes are in
fluid communication with each other when the device is rotated
about a rotational axis of the device; adding fluid to a plurality
of the first submicrovolumes; and rotating the device about the
rotational axis of the device to cause fluid in the first
submicrovolumes to be transferred to the second
submicrovolumes.
In another embodiment, a microfluidic device is provided that
comprises: a substrate; one or more microvolumes at least partially
defined by the substrate, each microvolume comprising a first
submicrovolume, a second submicrovolume where fluid in the first
submicrovolume is transported to the second submicrovolume when the
device is rotated about a first rotational axis, and a third
submicrovolume where fluid in the first submicrovolume is
transported to the third submicrovolume when the device is rotated
about a second, different rotational axis.
In another embodiment, a microfluidic device comprising: a
substrate; one or more microvolumes extending along a plane of the
substrate, each microvolume comprising a first submicrovolume, a
second submicrovolume where fluid in the first submicrovolume is
transported to the second submicrovolume when the device is rotated
about a first rotational axis that is positioned further away from
the second submicrovolume than the first submicrovolume, and a
third submicrovolume where fluid in the first submicrovolume is
transported to the third submicrovolume when the device is rotated
about a second, different rotational axis that is positioned
further away from the third submicrovolume than the first
submicrovolume.
In another embodiment, a microfluidic method is provided that
comprises: taking a microfluidic device comprising a substrate and
a plurality of microvolumes at least partially defined by the
substrate, each microvolume comprising an first submicrovolume, a
second submicrovolume where fluid in the first submicrovolume is
transported to the second submicrovolume when the device is rotated
about a first rotational axis, and a third submicrovolume where
fluid in the first submicrovolume is transported to the third
submicrovolume when the device is rotated about a second, different
rotational axis; adding fluid to the first submicrovolumes of the
microvolumes; and in any order rotating the device about the first
and second rotational axes to cause fluid from the first
submicrovolumes to be transferred to the second and third
submicrovolumes.
In another embodiment, a microfluidic device is provided that
comprises: a substrate; and a plurality of microvolumes at least
partially defined by the substrate, each microvolume comprising a
first submicrovolume and a second submicrovolume in fluid
communication with the first submicrovolume when the device is
rotated about a first rotational axis, wherein rotation of the
device about the first rotational axis causes a fixed volume to be
transported to each of the second submicrovolumes.
According to this embodiment, the plurality of microvolumes may
optionally further comprise one or more outlet submicrovolumes in
fluid communication with the first submicrovolume.
Also according to this embodiment, the plurality of microvolumes
may optionally further comprise one or more outlet submicrovolumes
where fluid in the first submicrovolume not transported to the
second submicrovolume when the device is rotated about a first
rotational axis is transported to one or more one or more outlet
submicrovolumes when the device is rotated about a second,
different rotational axis.
In another embodiment, a microfluidic device is provided that
comprises: a substrate; a first microvolume at least partially
defined by the substrate comprising a first submicrovolume; a
second submicrovolume where fluid in the first submicrovolume is
transported to the second submicrovolume when the device is rotated
about a first rotational axis; and a second microvolume at least
partially defined by the substrate comprising a third
submicrovolume; a fourth submicrovolume where fluid in the third
submicrovolume is transported to the fourth submicrovolume when the
device is rotated about the first rotational axis; and wherein
fluid in the second and fourth submicrovolumes are transported to a
fifth submicrovolume where the second and fourth submicrovolumes
are mixed when the device is rotated about a second, different
rotational axis.
According to this embodiment, the fifth submicrovolume may
optionally be in fluid communication with the second and fourth
submicrovolumes via the first and third submicrovolumes
respectively.
Also according to this embodiment, the device may further comprise
one or more outlet submicrovolumes in fluid communication with the
first and third submicrovolumes.
Also according to this embodiment, the device may further comprise
one or more outlet submicrovolumes in fluid communication with the
first and second submicrovolumes where fluid in the first and third
submicrovolumes not transported to the second and fourth
submicrovolumes when the device is rotated about the first
rotational axis is transported to one or more one or more outlet
submicrovolumes when the device is rotated about a third, different
rotational axis.
In yet another embodiment, a microfluidic method is provided that
comprises: taking a microfluidic device comprising a substrate, and
a plurality of microvolumes at least partially defined by the
substrate, each microvolume comprising a first submicrovolume and a
second submicrovolume in fluid communication with the first
submicrovolume; adding fluids to the first submicrovolumes; and
applying a centrifugal force to the device to cause a same volume
of fluid to be transported to the second microvolumes from the
first submicrovolumes.
Optionally, the microvolumes may further comprise an outlet
submicrovolume in fluid communication with the first
submicrovolumes. In such instances, the method may further comprise
transporting fluid in the first submicrovolume to the outlet
submicrovolume that was not transported to the second
submicrovolume when the centrifugal force was applied. The method
may also further comprise transporting fluid in the first
submicrovolume to the outlet submicrovolume that was not
transported to the second submicrovolume when the device is rotated
about a first rotational axis by rotating the device about a
second, different rotational axis.
In another embodiment, a microfluidic method is provided that
comprises: taking a microfluidic device comprising a substrate, a
first microvolume at least partially defined by the substrate
comprising a first submicrovolume and a second submicrovolume where
fluid in the first submicrovolume is transported to the second
submicrovolume when the device is rotated about a first rotational
axis, and a second microvolume at least partially defined by the
substrate comprising a third submicrovolume and a fourth
submicrovolume where fluid in the third submicrovolume is
transported to the fourth submicrovolume when the device is rotated
about the first rotational axis, the microvolumes further
comprising a fifth submicrovolume where fluid in the second and
fourth submicrovolumes are mixed when the device is rotated about a
second, different rotational axis; adding a first fluid to the
first submicrovolume and a second fluid to the third
submicrovolume; rotating the device about the first rotational axis
to transport the first and second fluids to the second and fourth
submicrovolumes; and rotating the device about the second
rotational axis to transport the first and second fluids from the
second and fourth submicrovolumes to the fifth submicrovolume.
In one variation, the fifth submicrovolume is in fluid
communication with the second and fourth submicrovolumes via the
first and third submicrovolumes respectively.
Optionally, the method further comprises removing fluid from the
first and third submicrovolumes that is not transported to the
second and fourth submicrovolumes prior to rotating the device
about the second rotational axis.
More specific examples of devices and methods according to these
numerous embodiments will now be described in relation to the
figures.
FIG. 13A illustrates a device for forming crystallizations by
rotation of the device, thereby applying centrifugal force. The
device 1300 comprises multiple crystallization wells 1301, each
having at least one inlet port 1302, a crystallization channel 1303
and an outlet port 1304. It is understood that during
centrifugation, the radially outermost port will, due to
centrifugal forces be the outlet port. However, for the purposes of
loading the cassette, either port 1302, 1304 may be used as an
inlet or outlet port. The device can have a centering device 1305
to center the device during centrifugation, or alternatively, the
device may be inserted in a receiver designed to mate with the
device.
As can be seen, the device is similar in design to a compact disc,
comprising a flat, circular plate of substrate with a hole in the
middle, preferably at the center of mass of the device.
Incorporated into the substrate is an array of crystallization
chambers. This design allows the crystallization agents to be added
to the device. Then, when the device is rotated, the
crystallization agents in the different chambers are each caused to
enter a corresponding crystallization well.
Given the symmetry of the design and the uniformity of the
centrifugal force that is applied, the design of the device
provides for a compact system where crystallization agents can be
first added and stored in the device. Then, when the device is
ready to be used, the device can be rotated to cause the prior
added crystallization agents to move within the device. As
illustrated in FIG. 13A, the rotational axis about which the
microfluidic device is rotated may be within a lateral footprint of
the device.
The design of the device also allows for multiple devices to be
stacked upon each other. This allows for a great number of devices
to be processed in parallel. FIG. 13B illustrates a plurality of
the devices shown in FIG. 13A where the devices are stacked
relative to each other when the centrifugal forces are applied so
that the same forces are applied to all of the devices.
FIG. 14A illustrates another embodiment of a device 1400 that is
designed to move fluids within the device by centrifugal force.
This design allows for the precise measurement of very small
volumes without the use of moving parts, electromotive force or
active pumps within the device. The device consists of at least two
inlet chambers 1401, 1401', a measurement channel 1402 for each
inlet, a waste channel 1403 from each inlet 1401 to a waste
reservoir 1404 or outlet, a mixing manifold 1405 connecting the
measurement channels 1402, 1402' and the crystallization chamber
1406. The manifold, can encompass the inlet port, or by pass it.
The measurement channels can be of identical or differing volumes,
dependent upon the need. The crystallization chamber can be of any
shape, shown here as either circular 1406 or rectangular 1406'.
Only one waste channel is illustrated, but each measurement channel
has an associated waste channel. These channels can be independent,
or by suitable design can form a manifold.
The device may be employed as follows; into each inlet chamber
1401, a volume of crystallization agent or substance to be
crystallized is added. The volume that is added does not need to be
precise or accurate. Instead, it is sufficient that the volume is
greater than a minimum volume for the measurement channel 1402. The
crystallization agents can be dispensed in advance of the substance
to be crystallized, enabling the device to be made in advance and
used as needed. Once the inlet chambers are all filled, the device
is rotated with the centrifugal force vector approximately aligned
as shown, for the loading spin (A).
The centrifugal force fills the measurement channel 1402
completely, leaving some residue in the inlet chamber 1401. A
subsequent measurement spin (B), removes the excess from the inlet
chamber and deposits the excess in the waste reservoir 1404 or
port, leaving the inlet chamber empty. At this point, the device
may be stored until needed. The inlet ports may be sealed by the
application of a tape, lid, septum, or by stacking the devices
together.
The rotational axis about which the microfluidic device illustrated
in FIG. 14A is rotated is positioned further away from the
measurement channel 1402 than the inlet chamber 1401. This provides
the centrifugal force vector the directionality that is shown
during the loading spin (A). The rotational axis about which the
microfluidic device is rotated may be within or outside of the
lateral footprint of the device.
The substance to be crystallized is then added into each inlet
chamber 1401'. The volume does not need to be precise or accurate.
Instead, it is sufficient that the volume be greater than a certain
minimum for the measurement channel 1402'. Once the inlet chambers
are all filled, the device is centrifuged with the centrifugal
force vector as shown, for the loading spin (A). This fills the
measurement channel 1402' completely, leaving some residue in the
inlet chamber 1401'.
A subsequent measurement spin (B) with the centrifugal force vector
approximately in this direction, removes the excess from the inlet
chamber and deposits the excess in the waste reservoir 1404 or
port, leaving the inlet chamber empty. At this point, the device
may again be stored until needed. The inlet ports may be sealed by
the application of a tape, lid, septum, or by stacking the plates
together.
It is noted that the rotational axis about which the microfluidic
device is rotated in the subsequent measurement spin (B) is
positioned in a different location than the rotational axis during
the load spin (A). In this instance, the different rotational axes
are laterally offset relative to each other. The rotational axis
for the measurement spin (B) is also positioned further away from
the waste reservoir 1404 or port than the inlet chamber 1401. This
positioning causes the centrifugal force vector to be in the
direction illustrated in regard to the measurement spin (B). It is
noted that the rotational axis about which the microfluidic device
is rotated during the measurement spin may also be within or
outside of the lateral footprint of the device. Although the
rotational axes are shown to be parallel and laterally offset
relative to each other, it should be recognized that the axes may
also be angled relative to each other.
Crystallization, or the test of crystallization is initiated by
centrifugation with the centrifugal force vector approximately in
the direction of the crystallization initiation spin (C). This
drives the crystallization agent or agents and the substance to be
crystallized through a mixing manifold 1405 into a crystallization
chamber 1406.
It is noted that the rotational axis about which the microfluidic
device is rotated during the crystallization initiation spin (C) is
positioned in a different location than during the load spin (A) or
the measurement spin (B). For example, the rotational axis for the
crystallization initiation spin (C) is positioned further away from
the inlet chamber 1401 than the measurement channel 1402. This
positioning causes the centrifugal force vector to be in the
direction illustrated in regard to the load spin (C), which in this
case is in the opposite direction than the centrifugal force vector
for the load spin (A). It is again noted that the rotational axis
about which the microfluidic device is rotated during the load spin
may also be within or outside of the lateral footprint of the
device.
The process illustrated in FIG. 14A is an example of a microfluidic
method that is provided by the present invention that comprises:
taking a microfluidic device comprising a substrate and a plurality
of microvolumes at least partially defined by the substrate, each
microvolume comprising an first submicrovolume, a second
submicrovolume where fluid in the first submicrovolume is
transported to the second submicrovolume when the device is rotated
about a first rotational axis, and a third submicrovolume where
fluid in the first submicrovolume is transported to the third
submicrovolume when the device is rotated about a second, different
rotational axis; adding fluid to the first submicrovolumes of the
microvolumes; and in any order rotating the device about the first
and second rotational axes to cause fluid from the first
submicrovolumes to be transferred to the second and third
submicrovolumes.
FIG. 14B illustrates how multiple devices, such as the device shown
in FIG. 14A may be processed together. As illustrated, the multiple
devices may be positioned radially about a rotational axis. During
each different load, measurement, and initiation spins, each device
may be positioned relative to the rotational axis so that the
corresponding vector is extending radially away from the rotational
axis. It should be noted that the multiple devices may
alternatively or in addition be stacked relative to each other, as
illustrated in FIG. 13B.
It is noted in regard to the various embodiments involving
centrifugal force, such as the embodiment shown in FIG. 14A, that
acceleration and deceleration, created by a change in a rate of
rotation of the device, can be used. In particular, when a device
is rotating and the rate of change of rotation of the device is
zero or close to zero, then the primary component of the force
vector is radial. However, when the device is initially at a
constant rotational speed, which could be zero, and then as the
rotational speed of the device is increased, the primary component
is initially orthogonal to the radius, and in the rotational plane,
tangential to the rotation. This is also true if the device is
decelerated. It is also noted that the faster the device is
accelerated, the larger the magnitude of the force.
This fact can be used to modulate the flow of liquid within the
microfluidic device. A large tangential force vector as the device
is being accelerated causes the liquid within device to initially
begin flowing in the counter to the direction of the initial force.
Thus, the inertial response of the fluid to the centripetal
acceleration is to appear to lag the acceleration of the device.
Similarly, if the device is decelerated, the fluid will lag the
deceleration.
This enables the production of devices that have different fluid
behavior depending upon the direction of rotation and the rate of
acceleration or deceleration.
In one example, described in relation to the device illustrated in
FIG. 14A, if the initial loading spin has a rotational axis on a
side of the device adjacent the crystallization chamber 1406 and
the device is rapidly accelerated clockwise, fluid placed in
loading port 1401 will flow into chamber 1402. Conversely, if the
device is initially rotated counter-clockwise with a rapid
acceleration, the initial force upon the liquid will direct the
liquid largely toward port 1403 and hence to the waste chamber
1404. If the device is accelerated slowly, the primary component of
the force vector acting upon the liquid will be radial and the
liquid will flow from the loading port 1401 into the chamber 1402,
regardless of the direction of rotation. Skilled artisans will
appreciate that by suitable design of the substrate and by
manipulation of the rate of change of the rotational speed, the
fluid flow in the device can be modulated to achieve very different
outcomes. It will be understood by those skilled in the art that
when the centrifugal force is neither parallel to nor normal to the
plane of the device, additional asymmetries in fluid flow may be
exploited for more complex fluid partitioning and combinations.
FIG. 15A illustrates how centrifugal force can be used to perform
precise measurements. FIG. 15A illustrates a repeating unit of the
centrifugal array in more detail. In this embodiment, one inlet for
crystallization agents 1501 and one inlet for a substance to be
crystallized 1501' are shown. Note that the lumens 1502, 1502'
connecting to the measurement lumens have a short neck near the
inlet chamber, orthogonal to the measurement spin. V represents the
measured volume after the measurement spin. During the measurement
spin, excess material in the inlet chamber, and the excess above V
is centrifugally ejected through lumens 1503, 1503' and hence
through lumens 1504, 1504' to the exit port or reservoir. Note that
lumens 1503, 1503', also have a narrow neck, initially oriented
parallel to and in the opposite direction to the loading spin
vector, ensuring that the liquids proceed down 1502 or 1502' to the
measurement lumens.
FIG. 15A thus illustrates a microfluidic method that comprises:
taking a microfluidic device comprising a substrate, and a
plurality of microvolumes at least partially defined by the
substrate, each microvolume comprising a first submicrovolume and a
second submicrovolume in fluid communication with the first
submicrovolume; adding fluids to the first submicrovolumes; and
applying a centrifugal force to the device to cause a same volume
of fluid to be transported to the second microvolumes from the
first submicrovolumes.
FIG. 15A thus also illustrates an embodiment of a microfluidic
device that comprises: a substrate; and a plurality of microvolumes
at least partially defined by the substrate, each microvolume
comprising a first submicrovolume and a second submicrovolume in
fluid communication with the first submicrovolume when the device
is rotated about a first rotational axis, wherein rotation of the
device about the first rotational axis causes a fixed volume to be
transported to each of the second submicrovolumes.
As illustrated, the plurality of microvolumes may optionally
further comprise one or more outlet submicrovolumes where fluid in
the first submicrovolume not transported to the second
submicrovolume when the device is rotated about a first rotational
axis is transported to one or more one or more outlet
submicrovolumes when the device is rotated about a second,
different rotational axis. When the microvolumes further comprise
an outlet submicrovolume in fluid communication with the first
submicrovolumes, the method may further comprise transporting fluid
in the first submicrovolume to the outlet submicrovolume that was
not transported to the second submicrovolume when the centrifugal
force was applied. The method may also further comprise
transporting fluid in the first submicrovolume to the outlet
submicrovolume that was not transported to the second
submicrovolume when the device is rotated about a first rotational
axis by rotating the device about a second, different rotational
axis.
FIGS. 15B-15G illustrate how centrifugal force can be used to
perform precise measurements and mixing. More specifically, these
figures illustrate a microfluidic device that comprises: a
substrate; a first microvolume at least partially defined by the
substrate comprising a first submicrovolume; a second
submicrovolume where fluid in the first submicrovolume is
transported to the second submicrovolume when the device is rotated
about a first rotational axis; and a second microvolume at least
partially defined by the substrate comprising a third
submicrovolume; a fourth submicrovolume where fluid in the third
submicrovolume is transported to the fourth submicrovolume when the
device is rotated about the first rotational axis; and wherein
fluid in the second and fourth submicrovolumes are transported to a
fifth submicrovolume where the second and fourth submicrovolumes
are mixed when the device is rotated about a second, different
rotational axis. As will be illustrated, the fifth submicrovolume
may optionally be in fluid communication with the second and fourth
submicrovolumes via the first and third submicrovolumes
respectively. As will also be illustrated, the device may further
comprise one or more outlet submicrovolumes in fluid communication
with the first and third submicrovolumes. As will also be
illustrated, the device may further comprise one or more outlet
submicrovolumes in fluid communication with the first and second
submicrovolumes where fluid in the first and third submicrovolumes
not transported to the second and fourth submicrovolumes when the
device is rotated about the first rotational axis is transported to
one or more one or more outlet submicrovolumes when the device is
rotated about a third, different rotational axis.
FIGS. 15B-15G also illustrate a method that comprises: taking a
microfluidic device comprising a substrate, a first microvolume at
least partially defined by the substrate comprising a first
submicrovolume and a second submicrovolume where fluid in the first
submicrovolume is transported to the second submicrovolume when the
device is rotated about a first rotational axis, and a second
microvolume at least partially defined by the substrate comprising
a third submicrovolume and a fourth submicrovolume where fluid in
the third submicrovolume is transported to the fourth
submicrovolume when the device is rotated about the first
rotational axis, the microvolumes further comprising a fifth
submicrovolume where fluid in the second and fourth submicrovolumes
are mixed when the device is rotated about a second, different
rotational axis; adding a first fluid to the first submicrovolume
and a second fluid to the third submicrovolume; rotating the device
about the first rotational axis to transport the first and second
fluids to the second and fourth submicrovolumes; and rotating the
device about the second rotational axis to transport the first and
second fluids from the second and fourth submicrovolumes to the
fifth submicrovolume.
It is noted that different sets and subsets of combinations
described herein can be performed without departing from the
present invention.
Referring to FIG. 15B a device is shown where a crystallization
agent 1507 has been added into the entry port, or well 1501. Also
shown is the material to be crystallized 1507' in a second entry
port or well. These materials need not be added
contemporaneously.
FIG. 15C illustrates the effect of centrifugal force on the samples
that were loaded to the device in FIG. 15B. Upon the application of
centrifugal force, the bulk of the material 1507 and 1507' proceeds
to fill the respective measurement lumens 1502 and 1502'. This
leaves some amount of excess material 1508 and 1508' in the initial
loading wells 1501 and 1501', respectively.
In FIG. 15D, the centrifugal force vector is changed such that the
force now directs the excess crystallization agent and excess
material to be crystallized 1508 and 1508' toward the waste ports
1504, 1504' via the respective waste lumens 1503, 1503'. After
applying this centrifugal force, the measurement channel is filled
with material to the point V in every measurement lumen.
FIG. 15E shows each lumen filled to point V, resulting in precise
volume measurements.
In FIG. 15F, the centrifugal force vector is again changed to align
in the direction shown. Centrifugal force in this direction drives
the crystallization agent 1507 and the material to be crystallized
1507' from the measurement lumens 1502, 1502', across the inlet
ports 1501, 1501' and through a manifold 1505, 1505' to the mixing
manifold 1506 and hence into the crystallization chamber 1500, as
the mixed material 1509.
FIG. 15G illustrates the final result, where the crystallization
chamber 1500 has been filled with the combination of the material
to be crystallized and the crystallization agent, or agents.
As will be appreciated, the process of making precise microfluidic
measurements and precise mixing by using centrifugal force can be
performed in a highly parallel manner, both by incorporating
numerous microvolumes into a given device, and by applying
centrifugal force to multiple different devices at the same time,
wherein the variations in acceleration, or deceleration, will be
uniformly applied over all devices and all lumens within said
devices.
6. Use of the Devices of the Present Invention to Determine Crystal
Growth Conditions
One of the intended uses of the devices of the present invention is
for improving the process of discovering novel crystal growth
conditions. By using the devices of the present invention, a
simultaneous, multiple factor approach can be implemented.
Current methods of vapor diffusion, hanging drop, sitting drop and
dialysis evaluate a single test condition in each instance. By
contrast, the present invention allows for multiple different
crystallization conditions to be created in the same lumen, thereby
allowing for multiple different crystallization conditions to be
tested. In some embodiments, gradients are created which create the
multiple different crystallization conditions. Diffusion of either
the sample being evaluated and/or the enclosing medium having a
viscosity such that the diffusion of the chemical moieties for
crystallization is much faster than the diffusion of bulk material
allows for the gradients to be created. This can be achieved either
through intrinsically viscous materials or additives such as
agarose, acrylamide, silica gel, or PEG, or by the use of filter
plugs, or by the use of enclosing channels that are sufficiently
thin in at least one dimension to limit macroscopic flow such that
diffusion of the chemical moieties for crystallization dominate.
These samples can be affected by sample droplets in a channel,
droplets within an enclosing crystallization medium, or
crystallization droplets or islands within an enclosing volume of
sample.
While the present invention is disclosed with reference to
preferred embodiments and examples detailed above, it is to be
understood that these examples are intended in an illustrative
rather than limiting sense, as it is contemplated that
modifications will readily occur to those skilled in the art, which
modifications will be within the spirit of the invention and the
scope of the appended claims. The patents, papers, and books cited
in this application are to be incorporated herein in their
entirety.
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